BIMM 100 Exam 1

covers LE 1-6, week 2-3 DI notes & slides, exam 1 review notes & slides (incl. practice problems), problem sets 1-3 (3rd is the optional problem set, but likely include flashcards on it)

def. gene

• 2 conditions for it to be the molecular basis of a gene

a unit of hereditary trait that shapes a specific trait of a cell or organism

• (hereditary traits - traits passed through generations)

_

DNA

• should be transmitted from cell-to-cell (or parent-to-child)

• should generate a trait

what did Griffith’s experiment discover? (1)

_

describe rough vs. smooth bacteria (← 2 each)

(& what happened to mice infected with each?)

_

what happens to:

• mice w/ smooth cell bacteria introduced to heat (←explain w/ 2)

• mice w/ smooth cell bacteria introduced to heat + rough cells (← explain w/ ~3, where 1 of them def. diff. b/w genes and a specific term)

_

are dead bacteria virulent or non-virulent?

_

genes shape __, while chromosomes do what? (1)

discovered that a trait, like bacterial virulence (cause death/disease), can be transferred from cell-to-cell

_

rough bacteria is non-virulent

• do NOT have polysaccharide “cloak”

• are destroyed by the mouse immune system

  • SO mice infected with R cells survive

smooth bacteria is virulent

• have polysaccharide “cloak”

• sneak past the mouse immune system

  • SO mice infected with S cells die

__

heat-killed smooth cells → mice survive

• b/c heat-killed (dead) smooth cells are non-virulent

  • heat kills bacteria

  • dead bacteria are non-virulent

dead smooth cells + live rough cells → mice die

• b/c live smooth cells are recovered from the dead mouse

  • dead cells can transfer their traits to live cells

  • ^ virulence can be transferred from cell-to-cell

• SO something else is transmitted from S cells → R cells in order to change the virulence of R cells, which are CHROMOSOMES

  • genes shape traits, while chromosomes carry genetic info & have both nucleic acids AND proteins

def. chromosomes

• describe (2)

___

label which is nucleic acid vs. amino acid

_

identify 5 parts of an amino acid

#of common amino acids

_

identify 3 parts of nucleic acid

name the 5 nucleotides (which 2 belong to what specifically)

are carriers of heritable traits/genes & are made of nucleic acids AND proteins

• when a cell divides, chromosomes are transmitted to both daughter cells

• inheritance of specific traits correlates to inheritance of chromosomes

__

amino acid has central carbon (alpha carbon), attached to:

• amino group (NH₂)

• side chain (R)

• carboxylic acid group (COOH)

• H

__

nucleic acid has multiple:

• phosphate group

• sugar ring (is deoxyribose sugar for DNA)

• Nitrogenous base

nucleotides (have one of each of the above)

• adenine, cytosine, guanine, thymine (DNA), uracil (RNA)

review: what molecule is needed to transmit S cells → R cells in order to change the virulence of R cells

conclusion of Avery-Macleod-McCarty experiment (1)

_

what happens to R cells if you:

• break open/lyse S cells, eliminate protein w/ proteinase, add R cells to S-cell lysate that lacks protein

• lyse S cells, eliminate DNA w/ DNase, add R cells to S-cell lysate that lacks DNA

chromosome, that contain nucleic acids (RNA, DNA) & proteins

• which led to hypothesis that nucleic acid or protein was the genetic molecule

_

conclusion:

• absence of DNA (the genetic material) will block rough/non-virulent cells from transforming into smooth/virulent cells (whereas, smooth cell DNA transforms rough cells into smooth cells)

• (aka smooth cell traits are transmitted to rough cells by DNA)

__

w/ proteinase

• S-cell lysate can still cause R/non-virulent cells to transform into S/virulent cells B/C the lysate doesn’t have protein BUT does have nucleic acid

• → protein is not the genetic material

w/ DNase

• R/non-virulent cells do NOT transform into S/virulent cells B/C the lysate does not contain DNA, even though it does contain RNA and proteins

• → DNA is the genetic material

include C1-5

_

DNA is 2 strands of __ in a helix

DNA is 2 strands of nucleotides in a helix

(picture should be deoxyribose sugar ring)

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(remember that a nucleotide is made up of 1 phosphate group, 1 sugar ring, 1 nitrogenous base)

(a nucleic acid is made of multiple nucleotides)

diff. b/w results & conclusion

results = data/ / observations

conclusion = interpretation

state what is attached at positions C1-3 and C5 in a pentose sugar ring

• for 1 of them, state its importance

C1’ — where nitrogenous base attaches (purine or pyrimidine base)

C2’ — determines if molecule is RNA or DNA (w/ hydroxyl group)

• 2’ OH means RNA (think RUH “ROH”)

• not 2’ OH, but 2’ H, means DNA

C3’ — where OH/hydroxyl group attaches, to form a polynucleotide strand (3’ OH)

C5’ — where phosphate group attaches

diff b/w purine vs. pyrimidine

which of the 5 bases are a part of each?

___

draw all the 5 bases (might help to think about the basic structure of purines & pyrimidines)

• & highlight which parts are hydrogen bond donors & acceptors

• ^ which 2 bases have 3 total donors/acceptors vs. 2

(MB know #s w/in a ring)

^^ can also refer to drawings in midterm notebook for better visualization

purine - 2 rings

• A, G

pyrimidine - 1 ring

• C, T, U

__

C-G have 3 H bonds

A-T have 3 H bonds

def. glycosidic bond

• enzyme that cleaves it

bond b/w C1’ of sugar ring & N-base

• cleaved by glycosylase

nucleoside vs. nucleotide (1 each)

• name 4 nucleosides

t/f: different #s of phosphates can compose a nucleotide (meaning?)

nucleoside has attached OH (hydroxyl) group

• adenosine, thymidine, guanidine, cytidine

nucleotide has attached phosphate group

(^ both have sugar ring & N-base)

__

true

• dAMP (deoxy””-5’-monophosphate), dADP (bi-), dATP (tri-)

name 4 things that make RNA diff. from DNA

RNA

• ribose sugar, where 2’ OH (think RUH “ROH”)

• has uracil, not thymine

• usually single stranded

• can function as an enzyme: “ribozyme”

def. phosphodiester bond

• what enzymes forms it (2) ← & name b/w what 2 things the bond forms between

• what enzymes cleave it (2)

this bond forms (1)

bond b/w 3’ OH & 5’ of 2 nucleotides (specifically b/w 2 sugar rings)

• (where 3’ OH becomes 3’ O that’s part of phosphate, which binds to the 5’ of another ring)

• 3” OH can be either with :

  • 5’ phosphate w/in the strand — DNA ligase

  • 5’ phosphate of incoming dNTP — DNA polymerase

__

made from DNA ligase & DNA polymerase

cleaved from exonuclease & endonuclease

__

phosphodiester bond forms the sugar-phosphate backbone w/in the SAME strand

describe 4 protein structures

• bond of secondary? structures (can only name 1)?

___

name if single vs. double-stranded nucleic acid chains

primary - linear polypeptide chain of amino acids

secondary - folding of backbone w/ H bonds

• alpha helix, etc.

tertiary - 3D folding of chain

quaternary - multiple chains into a protein

__

single - RNA

double - DNA

for discovering structure of DNA

brief description of Florence Bell vs. Linus Pauling model for DNA ← both were incorrect models of DNA

• what’s incorrect about Pauling’s model (2)

3 parts of correct DNA structure? ← by Franklin-Wilkins-Watson-Crick

direction of DNA movement

Bell — stacked nucleotides

Pauling — triple helix

• wrong about:

  • 3 strands of DNA in the helix (triple helix) — should be double-helix

  • bases being outside the helix — should have bases from opp. strands pair w/ e/o in the center of the helix

__

• 2 strands of DNA (double-helix)

• base pairs, where nucleotide bases on opposite strands form hydrogen bonds

• the 2 strands are anti-parallel to e/o (5’→3’, 3’→5’ which refer to the C in the sugar ring)

DNA moves from 5’ → 3’ end

can RNA be antiparallel? explain

yes

• even though it’s single-stranded, it can become a complex structure, where the single strand folds in on itself & the complementary bases can pair together ← anti-parallel

name the 2 types of intermolecular interactions present in DNA (& what are the interactions)

what about another one w/ what specific types of bonds?

__

(2) are important for stability of DNA

hydrogen bonds — base pairing (aka b/w bases of opposite strands)

Van der Waals forces — base stacking

• (does NOT include any of the 3 bonds talked about earlier)

_

covalent bonds

• glycosidic bonds

• phosphodiester bonds

__

base pairing & base stacking for stability

what 2 types of hydrogen bonds are for DNA (aka the molecules that base pair for DNA) vs. 1 type of H bond for proteins

DNA

• O — H(N)H

• N — H(N)

proteins

• O — H(N) ← for alpha helix, beta sheet

relation b/w H bonds & methyl group (CH3)

C

thymidine (“-side”)

__

hydrogen bonds aren’t usually formed w/ methyl groups (CH₃)

review(ish)

locate hydrogen bond acceptors vs. donors for 5 bases (probably draw it out)

__

t/f: molecules have additional H-bond donors and acceptors that aren’t highlighted by arrows

t/f: base pairs form due to alignment of hydrogen bond donors and acceptors

true

(i.e. the other H in NH₂)

_

true

the secondary structure of DNA and RNA, specifically (1), is sensitive to (2 ← specify a bit)

__

def. T_m

what happens as temp increases

• relate this to T_m (melting temperature) (2) ← & explain each

what happens as temp decreases

H bonds of secondary structure is sensitive to temperature & pH

• high temp & extreme pH (very high or very low pH), disrupt the secondary structure

_

T_m (melting temp)

• temperature where the helix is half double-stranded & half single-stranded (aka temp when 50% denatured)

_

as temp increase, the strands of DNA helix will denature/separate

• stable DNA helix = high Tm

  • ^ stable helix needs more heat to denature, so high Tm

  • (denatures 50% at higher temp, so more stable)

• unstable DNA helix = low Tm

  • ^ unstable helix needs less heat to denature, so low Tm

  • (denature 50% at lower temp, so more unstable)

as temp decreases, DNA strands w/ complementary sequences will anneal/zip up

t/f: DNA sequence also affects DNA stability & Tm of DNA (in addition to temp and pH)

• explain

true

• A/T and C/G have/form different #s of H bonds (2 vs. 3) and base-stacking interactions

• where C/G pair have a higher Tm

  • meaning C/G is more stable at higher temp than A/T which is more unstable at higher temp / more stable at low temp)

low pH is unstable

unstable helix is low Tm (needs less heat to denature)

buffer 1 has the lower Tm

3 conformations/forms of DNA in tertiary structure

which one allows nucleic acid bases & proteins to interact

__

function of quaternary structure

A-, B-, Z-DNA (have diff. configurations)

_

B-DNA conformation allows proteins to bind to specific DNA sequences

• this is essential for gene expression

____

quaternary structure of DNA helps in packing genomic DNA in cells

DNA is stored as __ in EUkaryotic cells

• describe

what about how DNA is packaged in PROkaryotic cells?

DNA is stored as chromatin in eu- cells

• DNA helix

• DNA helix + histones (are proteins) = nucleosomes

• nucleosomes are compacted into chromatin fiber

__

pro- genomic DNA is supercoiled

review:

state central dogma w/ processes (which process allows genetic info to be transmitted from parent cell to daughter cells via cell division)

genes are encoded in __

DNA → RNA → proteins

replication (of DNA), transcription (DNA ←→ RNA), translation (into protein)

• DNA replication allows genetic info to be transmitted from parent cell to daughter cells

__

genes are encoded in DNA

discovery from Meselson-Stahl experiment (1)

what serves as template to make daughter strand?

DNA replication is semi-conservative

• after replication, EACH helix has 1 strand of template parent DNA & 1 strand of newly synthesized daughter DNA

parent strand is the template for daughter strand

state key steps & enzymes involved in DNA replication (8 steps)

1. helicase denatures/unwinds DNA

2. topoisomerase relieves helix strain that occurs when unwinding DNA

3. RNA primase (aka primase) creates a short RNA primer to start DNA synthesis

4. DNA polymerase (DNA pol III) adds nucleotides via phosphoryl-transfer rxn

5. DNA pol “proofreads” via exonuclease activity

6. DNA pol (DNA pol I) removes RNA primer & replaces it w/ DNA

7. DNA ligase (aka ligase) joins together the DNA fragments by forming a phosphodiester bond

8. telomerase acts at the end of chromosomes to maintain their/the chromosome’s length

each human cell has ~__ feet of DNA, which takes ~__ hours for the human cell to replicate its genome b/c during mitosis, the cell copies the entire length of DNA before it divides

where does genome replication start (1)

• describe (1)

• in pro- vs. eukaryotes

~6 ft of DNA, ~8 hours

__

DNA replication starts at an origin of replication (“ori”)

• at an ori, 2 strands of DNA helix are denatured

pro- have single ori

eu- have multiple ori

why do eu- have multiple ori?

multiple ori allows for FAST genome replication

• (would take ~1 year for human cell to divide if human genome only had 1 origin of replication)

---

(OVERALL, not only regarding multiple ori, you want DNA replication to have a balance b/w being both fast & accurate)

function of helicase (~2), which causes __ that looks like __

• direction of movement

• forms (1 w/ 1)

helicases bind the ori & denature/unwind DNA by breaking the H-bonds, which causes tension that looks like supercoils

(locally denature DNA at the ori)

• move in opposite directions to unwind the helix & separate the strands

• forms the “replication bubble”, where there are 2 “replication forks” that move in opposite directions away from the ori

function of topoisomerase (1)

• function of SSB (is NOT an enzyme, but a protein)

as helicase unwinds DNA, topoisomerase relieves torsional stress that was caused by strand separation (prevent supercoils from forming)

__

SSB (single-strand binding protein ← not an enzyme)

• stabilizes single-stranded DNA (works together w/ topoisomerase in DNA replication)

function of primase (← which is a __ __)

def. (RNA) primers

• why is it needed?

RNA primase (an RNA polymerase)

• makes RNA primers based on DNA template

primers - short piece of DNA or RNA (DNA/RNA primer) that DNA polymerase needs to make DNA

• BUT we use RNA primers in genome replication

__

is needed b/c DNA polymerase (next step) CANNOT bind DNA and initiate DNA synthesis w/o a primer

replication forks move in what direction?

move in same direction as helicase moves aka same direction as DNA separating

state function of DNA polymerase, where it adds nucleotides (1 ← long)

daughter strands are made __

__

DNA pol. can only add nucleotides to the (1), so if it is the very beginning of genome replication w/o some synthesized part, what is needed?

• explain

• is the primer part of the immediate product (not talking abt final product)?

adds the complementary nucleotide to the template base

by forming phosphodiester bond b/w 3’ OH & 5’-phosphate / alpha-phosphate of incoming dNTP (polymerization, via phosphoryl transfer rxn)

^^^ (daughter strands are made simultaneously)

^^^ dNTP as in dATP, dGTP, dCTP, dTTP

__

DNA pol. can ONLY add nucleotides to the 3’ OH at the 3’ end of a primer (at the primer’s 3’ end)

• if starting new DNA strand (5’ → 3’), then need primase to form a primer, so that primase can add the first nucleotide to initiate a strand

primer is part of immediate product, located at the 5’ end of the synthesized DNA strand

t/f: DNA polymerase can’t initiate synthesis, but (RNA) primase can

true

• DNA polymerase can’t initiate synthesis, so it needs RNA primer

def. nucleoside analogs (2)

• function ← how?

give example & describe for (viruses):

• HIV infections

• Covid-19

describe for cancer

compounds that are structurally similar to nucleosides & that are used to combat DNA & RNA viruses and cancer

• block replication of viral genomes

  • nucleoside analog is incorporated into the strand during DNA (or RNA) synthesis, but the analog stops further synthesis which prevents viral replication

__

ex:

• AZT treats/combats HIV infections, where AZT is an analog of the nucleoside thymidine

• b/c thymidine has 3’ OH for adding nucleotides during DNA synthesis, but AZT does NOT have 3’ OH so no nucleotides added → DNA synthesis stops

  • AZT inhibits viral replication

ex:

• Remdesivir combats Covid-19, where Remdesivir is an adenosine analog

__

slow mitosis & proliferation of cancerous cells

how are RNA primers removed & replaced? (& by what?)

DNA polymerase 1 (DNA pol I) removes RNA primers & fills in the gap w/ DNA

• (aka simultaneously removes the primer from the 5’ end of newly synthesized strand & replaces it w/ DNA)

---

(DNA pol binds at the 3’ end of the RNA primer & moves in the direction of 5’ → 3’)

function of DNA ligase

seals the gap by forming a phosphodiester bond

• (the gap is b/w where DNA pol filled in the gap & part of synthesized strand)

• (aka when the primer is replaced by DNA, a new piece of DNA is made BUT that piece is not attached to the neighboring DNA, which creates a gap b/w the 3’ & 5’ ends of neighboring pieces)

name these 2 synthesized daughter strands that: (& def. ← 2 each)

during genome replication, the 2 synthesized daughter strands (that are anti-parallel) in the 5’ → 3’ direction (b/c only direction DNA pol. moves in)

^ these 2 daughter stranded are synthesized __

leading strand (aka leading daughter strand)

• is made in 1 piece, using only 1 (RNA) primer

• made in the same direction that the replication fork moves

lagging strand (aka lagging daughter strand)

• is made in pieces (Okazaki fragments), using multiple (RNA) primers

• made in the opposite direction of the replication fork

__

simultaneously

also, which part would be the chromosome end (aka __)

telomere (aka chromosome end) is the end where the 2 parent strands haven’t unwound/separated/denatured yet (aka where parent strands are still together/intact)

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for the problem in pic:

you can identify the 5’ and 3’ end from the direction of the synthesized DNA daughter strands that move in 5’ → 3’ direction, SO the ends of parent strands would the opposite of the synthesized strands (b/c anti-parallel)

you can identify the leading vs. lagging strand by splitting the diagram in half vertically

• the blue move in same direction as fork

• the red move in opp. direction from fork

at the ends of linear chromosomes (aka at telomeres), what happens if nothing after ligase happens & you continue DNA replication?

• there is a gap (b/w 3’ and 5’ of neighboring ends), but (the leading strand has no gap)

• for lagging strand synthesis:

  • primase adds an RNA primer at 5’ end of chromosome

  • when primer is removed, there’s a gap at the end of the new chromosome

• ligase joins the 2 Okazaki fragments w/ phosphodiester bonds

there is a gap at the end of the telomere WHERE chromosomes get shorter each time IF you don’t fill in the gap and just continue genome/DNA replication

function of telomere (1), which is a __ polymerase

telomerase is a __ __ (← term relates to transcription)

_

after telomerase performs its role, what happens to the lagging daughter strand?

telomerase (a DNA polymerase)

• extends the 3’ end of the parent strand, using a RNA template, to maintain chromosome length

telomerase is a reverse transcriptase

• (b/c makes DNA based on an RNA template, where central dogma is DNA → RNA → protein)

_

then, the lagging daughter strand extends via primase + DNA pol + ligase using this newly synthesized parent strand as the template

multiple ori allows for fast genome replication, but this can lead to (1)

you want …

fast replication can lead to mistakes in DNA replication, like mismatches

instead, you want a balance b/w replicating a genome quickly & accurately (i.e. making only 2-3 mistakes)

def. mutation

• can be caused by (1) or (1)

• can be either (3)

ANY change in DNA sequence of a cell

• caused by mistakes during cell division (replication) OR exposure to DNA-damaging agents in the environment

• can be harmful, beneficial, or have no effect (neutral)

just state the 2 mechanisms of repairing mismatches during DNA replication (1 of them also has a specific term)

which is immediate vs. not

• exonuclease activity by DNA polymerase (“proofreading”)

  • immediate / during replication

• mismatch repair

  • not immediate / after replication is done

t/f: different forms of nucleotide bases can result in mismatches (“errors”) during DNA synthesis

• explain generally

• then, explain w/ ex. of cytosine

how do these spontaneous ““ shifts in the base occur? they can cause more __ nucleotides

true

• nucleotide bases can exist in 2 forms called tautomers (aka shifting of protons), where 1 form is more stable than the other: predominant, major vs. minor form

• the minor and major forms have different patterns of H bond donors and acceptors

__

• cytosine has a major & minor form

• the minor form of C can pair w/ A (adenine), but the major form cannot & only binds to G (guanine)

  • this C/A can lead to a change in the DNA sequence

_

these spontaneous proton shifts in the base can cause more mismatched nucleotides

• occur by:

  • minor form is paired to the template (w/ the complementary nucleotide base)

  • before DNA polymerase continues to add nucleotides after this, the minor base changes back to the major form B/C the minor form is unstable

  • this would be a spontaneous mismatch

what are the 2 enzyme activities of DNA pol? what bond is affected?

• specific term for 1 of them

(^^ this question is not completely referring to how to repair mismatch)

• polymerization, where DNA pol adds nucleotides via polymerase domain (Pol)

• exonuclease activity, where DNA pol removes mismatched nucleotides via exonuclease domain (Exo)

  • “proofreading”

^ both affect phosphodiester bonds (make vs. break, respectively)

---

(can refer to past flashcard for pic of polymerization, via phosphoryl-transfer rxn)

DNA pol adds/removes nucleotides to/from the __ end

3’ end

(i.e. DNA pol adds nucleotide at the 3’ OH of prior neighboring sequence, in the direction of 5’ to 3’)

DNA polymerase is what type of exonuclease & what type of polymerase?

• function of both

DNA polymerase I is what type of exonuclease & what type of polymerase?

• function of both

DNA pol

• 5’-to-3’ polymerase (adds DNA)

  • start at 3’ end

• 3’-to-5’ exonuclease / 3’ exonuclease (“proofreading” — remove incorrectly added bases, like mismatch and insertion/deletion)

  • starts acting at the 3’ end

DNA pol I

• 5’-to-3’ polymerase (adds DNA)

  • start at 3’ end

• 5’-to-3’ exonuclease / 5’ exonuclease (remove RNA primer)

  • starts acting at the 5’ end

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(the green and purple ovals are the Exo / exonuclease domain)

how would DNA pol recognize & remove mismatched nucleotides, based on the picture

DNA pol recognize mismatched base b/c 3’ OH of cytosine is not positioned properly

DNA pol then uses its 3’-to-5’ exonuclease activity to remove the mismatched base

exonuclease activity by DNA pol starts when there’s a spontaneous tautomeric shift that stops continuation of DNA synthesis,

so why doesn’t mismatch repair stop DNA synthesis when there’s a mismatch?

DNA synthesis continues b/c the mismatched nucleotide was not recognized

• (mismatch repair occurs after DNA synthesis is done b/c there’s no proofreading)

while exonucl. activity by DNA pol is proofreading that occurs during DNA synthesis

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exonuclease activity/proofreading occurs during DNA synthesis

mismatch repair (MMR) occurs after end of DNA synthesis

BER and NER occurs after the end of DNA replication

name & def. 2 mismatch repair enzymes

• 1 of them can act __ or __

_

review(ish):

another name for each (& function of each):

• 3’-to-5’ exonuclease

• 5’-to-3’ exonuclease

endonuclease — cleaves phosphodiester bonds w/in a strand

• can act EITHER upstream (at 5’ end) OR downstream (at 3’ end) of the mismatch (cleaving ONE phosphodiester bond in mismatch repair)

exonuclease — cleaves phosphodiester bonds at the end of a DNA strand

• EITHER upstream OR downstream

(^^ both cleave phosphodiester bonds, just at different positions in a DNA strand)

__

3’-to-5’ exonuclease (aka 3’ exonuclease) (aka 3’-5’ ““)

• proofreading / remove mismatch

5’-to-3’ exonuclease (aka 5’ exonuclease) (aka 5’-3’ ““)

• remove RNA primer

which DNA strand do endonucleases cleave?

• how do they know?

  • why is it like this?

mismatch repair machinery cleaves the non-methylated DNA

• the mismatch repair machinery can identify the parent strand b/c it is methylated (CH₃), while the daughter strand is not methylated

  • over time, genomic DNA accumulates methylation (parent strand is old, while daughter strand is new)

__

picture:

No b/c methylation at this position doesn’t disrupt the hydrogen bonds that form b/w adenine & thymine (A-T base pair is intact)

how do they both work together in mismatch repair:

• endonuclease, exonuclease, mismatch repair machinery, helicase, ligase, DNA pol (← is stated out of order)

^^ aka steps of mismatch repair (5)

_

& include which specific exonuclease is used depending on relative positions of two specific things (← 2)

• repair machinery/proteins recognize the mismatch & identify the unmethylated daughter strand

• endonuc. makes a “nick”/cut ONE phosphodiester bond near the mismatch

  • (nicks forms “near” aka EITHER upstream OR downstream of the mismatch, but not “at” the mismatch)

• helicase unwinds the DNA for exonuclease to access the DNA

• exonuc. removes nucleotides past the mismatch (aka including and after the mismatch), EITHER - OR -:

  • 5’ (5’ to 3’) exonuclease IF “nick” is 5’ / upstream from the mismatch

    • so moving from nick to mismatch is downstream (5’→3’)

  • 3’ (3’ to 5’) exonuclease IF “nick” is 3’ / downstream from the mismatch

    • so moving from nick to mismatch is upstream (3’→5’)

• DNA pol synthesizes new NDA

• ligase joins the 3’ OH & 5’ phosphate of neighboring nucleotide w/in the same strand (fills in the gap) in newly synthesized daughter strand

^^ endonuclease can cleave either upstream (5’ → 3’) or downstream (3’ → 5’)

name 2 of various ways that double-stranded breaks (DSBs) occur

_

name 2 ways that double-stranded DNA breaks (DSBs) can be repaired

• which is error-prone vs. precise repair? why?

• exposure to environmental mutagens

• deliberate breaks caused during gene editing (aka gene editing machinery) (will go over in later unit)

__

double-stranded DNA breaks can be repaired via:

• end-joining (EJ)

  • error-prone (b/c mutations are introduced a lot at the break)

• homology-directed repair (HDR), such as homologous recombination

  • precise repair (b/c homologous sequences act as the template for precise repair of the break)

briefly state what happens in end-joining vs. homologous recombination (1 each)

& again, explain why it’s error-prone or precise repair

end-joining

• broken ends are joined together

^^ during EJ, nucleotides might be added or removed around the break → changing the DNA sequence

__

homologous recombination

• new DNA is made to repair the break

^^ a similar/homologous sequence acts as the template sequence for repairing the break

explain the process of end-joining (for double-stranded break repair) (3)

• a protein complex (Ku + DNA-PK) binds to the broken ends & bring the ends together

• broken ends are modified (in varied ways) that change the DNA sequence

  • (aka usually have mutations at the site of the break)

• DNA ligase makes sure that the broken DNA is re-sealed

__

^^ the modifications are variable (i.e. exonuclease often removes a variable # of nucleotides from the ends) → SO variations in end-repair

briefly describe the 2 DNA helices involved in homologous recombination

__

where can a cell find a homologous sequence to repair the DSB? (2 w/ explanation)

• a DNA helix w/ 2 broken strands

• a DNA helix w/ a homologous sequence to the double-stranded broken helix

__

find homo- sequence via:

• sister chromatid — human cells are diploid, meaning they have 2 copies of each chromosome (1 from each parent) SO chromosome pairs are homologous chromosomes

• exogenous DNA — DNA w/ homologous sequence can be injected into cells during gene editing

answer the Q

& what does it result in/create? (1)

draw the 3’/5’ ends & the 5’ exonuclease with arrows of direction its moving in

5’ exonuclease — removes nucleotides from the 5’ end of one strand

• SO that the 3’ end of the opposite strand is single-stranded → 3’ overhang

---

(red shows 3’ overhangs)

explain the process/steps of homologous recombination (for double-stranded break repair) (6)

• in the helix w/ DSB, 5’ exonuclease cleaves the nucleotides at the 5’ end of one strand → make 3’ single-stranded ends on the opposite strand aka 3’ overhang

• 3’ overhang invades the helix w/ the homologous sequence

• DNA pol. extends the 3’ end of this invading strand UNTIL the displaced strand (of homo- sequence) base pairs w/ the OTHER 3’ overhang (to cover the break)

  • (3’ overhang acts a primer for DNA pol., except it is part of the DNA sequence)

• DNA pol. extends this other 3’ end (to cover the break)

• DNA ligase seals the two gaps → to form Holliday structures (“X”)

• the 2 Holliday structures are resolved → get repaired DNA

  • (in 2 ways: cut → non-crossover & cut → crossover) ← don’t have to know

why is it important to form the 3’ overhangs (1) & explain

the 3’ overhang brings in DNA polymerase

• b/c the 3’ overhang has a free 3’ OH, that DNA pol can add nucleotides to (b/c DNA pol starts adding nucleotides at the 3’ end)

^ this is also why you don’t need RNA primase in homologous recombination

review:

which specific exonuclease is used depending on relative positions of nick & mismatch (← 2)

__

review:

which removes single mismatch vs. large nucleotide sequence that can contain multiple mismatches: exonuclease activity via DNA pol OR mismatch repair

• 5’ (5’ to 3’) exonuclease IF “nick” is 5’ / upstream from the mismatch

  • so moving from nick to mismatch is downstream (5’→3’)

• 3’ (3’ to 5’) exonuclease IF “nick” is 3’ / downstream from the mismatch

  • so moving from nick to mismatch is upstream (3’→5’)

^^ just depends on relative position of nick and mismatch

can act EITHER upstream or downstream of the nick, but not in both directions at the same time

__

removes single mismatch — exonuclease activity via DNA poly

• (b/c acts once it recognize a mismatch during replication)

large nucleotide sequence that can contain multiple mismatches — mismatch repair

• (b/c acts after replication is over, so can possibly have multiple mismatches that are removed by the exonuclease)

what 2 enzymes catalyze the formation of phosphodiester bonds?

• difference b/w the 2 things that the bond forms between

DNA ligase

• b/w 3’ OH and 5’ phosphate w/in a strand (that already exist, which is why they’re w/in the strand) of 2 different nucleotides w/in a strand

DNA polymerase

• b/w 3’ OH & 5’ phosphate of incoming dNTP of 2 different nucleotides w/in a strand

t/f: primase (a type of RNA polymerase) can initiate DNA synthesis de novo

• what does “de novo” mean?

true

• “de novo” — from the beginning

just notice that:

• one side of ori is the synthesized leading strand, other side of ori is the synthesized lagging strand

• DNA always synthesized from 5’ → 3’

• leading or lagging strand is deter. by its synthesis relative to the direction the replication fork moves in

just notice that:

name & def. 3 types of mutagenic chemical modifications that affect the DNA base

(^ aka changes to nucleotide bases can cause mutations if not repaired via DNA repair mechanisms)

alkylation — addition of methyl or ethyl that disrupts a H-bond from forming (can’t form H bond aka disrupts base pairing)

• (methylation w/ CH₃ or ethylation w/ CH₂CH₃)

deamination — remove amine group & replace w/ carbonyl group

• (replace “—NH2” w/ “=O”)

depurination — remove the entire purine base

is methylation of guanine O6 mutagenic? (may help to draw picture)

• what happens to base pairing if methylated guanine O6?

when is methylation mutagenic?

_

diff. b/w methylation & alkylation (revew-ish)

yes b/c prevents it from being a H-bond acceptor

• instead, O6-methylguanine pairs with thymine (G^6-me-T, instead of G-C)

__

methylation is mutagenic if it occurs at a position that disrupts hydrogen bonds b/w standard base pairs

__

methylation — only adds a methyl group

alkylation — can add either a methyl or ethyl group

try to draw out adenosine w/o looking at picture & figure it out

for (deoxy)adenosine, is methylation at the circled positions N1, N6, and N7 mutagenic or not? explain

at N1: mutagenic

• N1 is acceptor in a base pair w/ T (thymine)

at N6: not mutagenic

• N6 is donor in a base pair w/ T, BUT N6 has 2 H’s (b/c NH₂)

at N7: not mutagenic

• even though N7 is a hydrogen bond acceptor, it is NOT involved in base pairing

• aka N7 is not a H-bond acceptor in a standard base pair

(review that deamination is removing amine group & replacing it w/ carbonyl group)

t/f: deamination of a base can change the base’s identity

__

deamination occurs __, but can also be catalyzed by __

__

deamination of C will convert C into __

• which makes the standard base pair of (1) into (1) mismatch, which can lead to mutations ← usually the most common mutation from this specific mismatch is called (1)

what may explain why DNA lacks uracil (U)?

true

__

deamination occurs spontaneously, but can also be catalyzed by enzymes

_

deamination of C: _regularC (cytosine) → U (uracil)

• standard base pair of C:G into U:G mismatch

• U:G mismatch can cause mutations, most commonly the C>T mutation (aka C → T mutation)

  • (C>T mutation goes from standard base pair of C:G → T:A)

__

DNA may lack uracil b/c of spontaneous deamination of cytosine

what base cannot be deaminated?

__

base-deamination changes these bases into what:

• cytosine

• 5-methylcytosine

• adenine

• guanine

& know the structure of 5-methylcytosine (draw it)

thymine can’t be deaminated b/c thymine has no amine group to replace w/ carbonyl group

__

C → U

C^5-me → thymine

A → hypoxanthine

G → xanthine

review:

def. of depurination

• what bases are affected

• def. apurinic

• what bond is cleaved/removed?

remove entire purine base (adenine, guanine)

• cleaves the glycosidic bond

_

apurinic — no purine base b/c it has been removed

• (NOT apurinic if it has a pyrimidine base)

alkylation, deamination, depurination were all mutagenic CHEMICAL modifications, but

what is the mutagenic STRUCTURAL modification (1 ← name & def.)

• 2 affected bases

• def. kink

• usually forms b/w …

pyrimidine dimers

• UV light causes a kink to form B/C of covalent bond that is made b/w pyrimidines on the same strand

  • kink — structural change to DNA helix

• affects pyrimidine bases: thymine, cytosine

  • NOT usually uracil

  • SO like covalent bond b/w T-T on same strand

• usually forms b/w T-T

just name 3 DNA repair mechanisms (that repair damaged base(s) that occur independently from replication)

base excision repair (BER)

nucleotide excision repair (NER)

direct repair

def. direct repair

is not a pathway for DNA repair, but is instead, a class of different enzymes that change/modify a base

(i.e. methyltransferase does demethylation/remove methyl group to revert the mutagenic methylation of O6 → into normal/unmethylated guanine)

how do you prevent a mistake from becoming a mutation?

repair machinery must act before the cell divides

aka

DNA repair must happen before replication happens, or else the mistake will become a PERMANENT mutation (after replication, mismatch becomes a mutation)

def. base excision repair (BER)

• explain w/ example of: BER steps for deaminated 5-methylcytosine aka guanine (4)

repairs that change DNA at ONE or few nucleotides & do NOT cause a major distortion of DNA

(remove 1 or few damaged/mismatched bases before replication occurs)

__

BER w/ deaminated 5-methylcytosine aka guanine:

• DNA glycosylase cleaves the glycosidic bond (b/w the sugar ring & base) —→ to make abasic nucleotide (aka nucleotide w/o base)

  • glycosylase is NOT specific in which strand’s mismatched base is changed (aka recognizes the mismatch/damage, but not the strand)

• AP endonuclease cleaves the phosphodiester bonds to remove the abasic nucleotide w/in the strand → forms a gap

  • (AP endonuc. is either apurinic or apyrimidinic endonuclease)

• DNA polymerase synthesizes new DNA to fill in gap

• DNA ligase seals the remaining nick

is DNA glycosylase need to repair depurinated bases? why? what would happen as the first step?

no

• b/c depurinated bases don’t have a base, meaning that there is no glycosidic bond (which usually forms b/w sugar ring + base)

• so glycosylase (which cleaves glycosidic bonds) is not needed for depurinated bases

SO BER’s the first step would be AP endonuclease (skip the glycosylase step)

def. nucleotide excision repair (NER)

• name example of NER

explain steps of NER using pyrimidine dimers

repairs that change DNA at multiple nucleotides & causes a major distortion of DNA

• like pyrimidine dimers

__

NER using pyrimidine dimers:

• recognize damage (aka pyrimidine dimers, like T-T)

• helicase opens/unwinds the helix

• endonucleases remove multiple nucleotides surrounding the damaged region (on the same strand)

  • specifically, excision endonucleases aka excinucleases, like XP-F and XP-G

• DNA polymerase synthesizes new DNA to fill in the gap

• DNA ligase seals the remaining nick

explain how (regular/unmethylated) C deamination in dividing cells → results in base-pair mismatch & in a C>T mutation (3 steps)

• & state transition of standard & non-standard/mismatched base pairs @ each step

• (unmethylated) C is deaminated into U

  • so goes from C:G → U:G

• cell division/replication occurs (dividing cells), where U is though to be part of the parent strand, SO DNA pol. pairs it w/ A in newly synthesized daughter strand

  • aka U:G —(cell div./repl. using U strand as the parent template strand)→ U:A

• after cell division is done, BER occurs to replace U w/ T (B/C it recognizes that uracil is not apart of DNA)

  • so U:A → T:A

(C:G → U:G → U:A → T:A ————— this is a C>T mutation)

^^^ b/c replication happened when mismatch was still present/not fixed, it caused a (permanent) mutation

explain how 5-methycytosine deamination in dividing cells → results in mismatch & in mutation

• deamination of C^5-me, where C^5-me:G → T:G

• cell division/replication occurs (dividing cells), where makes 2 daughter strands: 1 mutant T:A & 1 wild-type C:G strand

  • mutant strand is T:A (b/c has diff. base pair from original starting, standard base pair)

  • wild-type strand is C:G (b/c has same base pair as original starting base pair)

(C>T mutation in mutant strand)

explain how 5-methycytosine deamination in NON-DIVIDING cells → results in mutation

specifically results in C>T mutation

• b/c deamination of C^5-me, where C^5-me:G → T:G

  • (aka C^5-me → T)

can a mutation (like a C>T mutation) occur from:

• deamination of C in dividing cell

• deamination of 5-methylcytosine in non-dividing cell

• deamination of C in non-dividing cell

• deamination of 5-methylcytosine in dividing cell

deamination of C in dividing cell

• YES, b/c C:G (when deamination)→ U:G → U:A (when cell division/replication) → T:A (when BER of T)

deamination of 5-methylcytosine in non-dividing cell

• likely YES

  • b/c C^5-me:G (when deamination) → T:G

    • aka C turns into T, so is C>T mutation

deamination of C in non-dividing cell

• likely NOT

  • b/c C:G (deamination) → U:G (BER) → original C:G

    • b/c BER recognizes that U is not part of DNA

    • this happens despite the glycosylase step of BER doing non-specific cleaving of a strand, BER will recognize that U is usually part of RNA, not DNA

deamination of 5-methylcytosine in dividing cell

• YES, b/c C^5-me:G (when deamination) → T:G (when replication) → get two daughter strands of T:A & C:G

  • mutant strand of C>T mutation is T:A (b/c causes a change in/is different from the starting standard base pair)

  • wild-type DNA strand is C:G (b/c is the same base pair as the original standard base pair)

^^^

if mismatch/mutation is NOT repaired before a cell divides, it will cause a (permanent) mutation in the parent strand & in synthesized daughter strand

yes b/c there’s no 2’ OH (think RUH “ROH”), so that means it’s DNA

• b/c DNA vs. RNA is deter. by the 2’ sugar ring, not the type of base (i.e. not U vs. T)

for 5-methylcytosine deamination in non-dividing cells (shown in picture),

how does the repair machinery deter. which nucleotide is the original vs. mutant?

it can’t

b/c the “mutant” deaminated 5-methylcytosine is T (thymine)

BER occurs as the next step after deamination (b/c non-dividing cells):

• sometimes BER removes T → then restored to original base pair of C:G

• sometimes BER removes G → then sequence will mutate/change into T:A

t/f: spontaneous deamination of cytosine changes our genome & results in C>T mutations

• # of cytosines spontaneously deaminated per day

• % of human genome that is made of cytosine

__

t/f: C>T mutations are highly represented in “mutational signatures” associated w/ cancer

_

t/f: malfunctioning DNA repair machinery leads to predisposition to disease

true

• 100-500 Cs deaminated / day

• 20% of human genome that is made of cytosine

__

true

• b/c cancers are actively dividing cells

__

true

human genome has (#) base pairs

t/f: no two genomes have identical sequences b/c our genomes have many SNPs / single-nucleotide polymorphisms

t/f: a mutation may not even be detected b/c there may not be a “correct”/common nucleotide for that position in the genome

3.2 billion base pairs

_

true

true

incorrect base incorporation during DNA replication is fixed how?

__

chemical changes to DNA bases (alkylation, deamination, depurination) is fixed by (← state for each of the 3)

structural changes to DNA (pyrimidine dimers) is fixed by (1)

fixed by DNA polymerase’s exonuclease activity OR mismatch repair

__

chemical changes to DNA bases (alkylation, deamination, depurination) is fixed by:

• alkylation — by BER or direct repair (methyltransferase)

• deamination — by BER (need glycosylase)

• depurination — by BER (don’t need glycosylase)

structural changes to DNA (pyrimidine dimers) is fixed by — NER or direct repair

early discoveries from mapping genes (~4)

• first genetic map of chromosome (Sturtevant)

• DNA is the genetic molecule (…McCarty experiment)

• pieces of DNA “hop” around the genome, which disrupts genes & changes traits

• structure of DNA solved

for study on location genes w/in a genome:

• explain experiment w/ pigmentation of corn

kernel color (trait), corn/maize genome

• genomic DNA (chromosomes) are treated w/ DNA stain to get patterns of light and dark bands → used to find parts of genome that contain genes correlating w/ a specific trait

  • when the genomic fragment Ds that jumps around is in a position → purple kernels

  • when genomic fragment Ds is in another position → colorless/yellow kernels

(the genomic fragment Ds is the transposon)

def. Mendelian inheritance

__

def. transposons

• name 2 broad classes of transposons & briefly def.

t/f: more than half the human genome s made of transposable elements

genes are transmitted in a constant, predictable way from parent-to-child

__

transposons — pieces of genomic DNA that can “jump” around (aka can be pasted/inserted into different positions of a genome to affect the expressed traits)

• DNA transposon — cut & paste

• retrotransposon — copy & paste

__

true

describe how DNA transposons work (incl. enzymes involved) (2)

DNA transposons

• transposase cut/excises DNA transposon out of the original position in genome/DNA sequence (& flanking DNA leaves) → make a DNA intermediate

• transposase pastes DNA inter. into another part of the genome (the target DNA) → make a transposed mobile element, where original/same DNA transposon is in different position

describe how retrotransposons work (incl. enzymes involved) (3)

__

how many copies of the retrotransposon do you have at the end?

_

def. reverse transcriptase (give an example)

retrotransposons

• RNA polymerase copies retrotransposon into RNA (& original sequence w/ retrotransposon + flanking DNA leaves ← no excision of original retrotransposon) → make a RNA intermediate

• via reverse transcriptase: RNA sequence is reverse transcribed from RNA into DNA → make a DNA intermediate

• DNA inter. is pasted into another part of the genome (the target DNA) → make a transposed mobile element, where copy of retrotransposon is in different position

__

2 copies of retrotransposon: original (“donor DNA”) & newly synthesized transposon (“transposed mobile element”)

---

reverse transcriptase is a DNA polymerase that uses an RNA template

• like telomerase

cont. kernel pigmentation study

the movement of a transposon in the maize genome causes variation in the (1)

---

t/f: insertion of transposon can have the effects of decreasing OR increasing gene expression (i.e. pigment). ALSO, the presence and absence of transposons can also affect the expression of the trait

_

t/f: transposons induce variations in traits, which can include intermediate expressions of the trait

_

t/f: transposons induce variation in traits that contribute to evolution

kernel pigmentation

• b/c insertion of transposons in new positions w/in genome causes variations in expressed traits

• (aka transposons induce variations in traits)

__

true

• w/ inserted transposon → either decrease or increased expression of trait

• w/ or w/o inserted transposon → either decrease or increased expression of trait

__

true

• i.e. no transposon is black grapes, w/ transposon is white grapes, partially removed/piece of transposon is red grapes

_

true

• i.e. rise in industry caused moths to evolve from peppered to black appearance

where are genes located in the genome (2 parts)

genes are present in consistent, predictable positions (fixed positions) w/in genome of an organism

BUT some pieces of DNA called transposons can jump in & out of genes (insertion + excision), which affects traits

t/f: gene structure is sim. in all eukaryotic genomes

name & def. 3 common parts of a gene in eu-

• what does 1 of them tell us (2)

true

__

enhancer

• part of DNA sequence that binds proteins, like TFs, that stabilize or destabilize the RNA polymerase bound to the promoter

promoter

• part of DNA sequence that binds RNA polymerase, SO when stably bound, starts RNA transcription

transcription unit

• part of DNA sequence that encodes for RNA (aka DNA template for RNA synthesis / DNA → RNA)

  • where if RNA codes for → proteins → trait, then that transcription unit is part of the “blueprint”

  • but can also have RNA encoding for→ trait

__

enhancer tells us where a gene is expressed & the amount of RNA transcribed

name & def. two elements/parts of mRNAs / of transcription unit

exons — part of mRNA that is transcribed & kept in mature mRNA

introns — is removed from mRNA

what happens if transposon is inserted/pasted into an enhancer?

• gene is expressed in different areas/different tissues (← pattern)

• amount of RNA expression (from RNA transcription) & amount of protein changes

BUT

• protein made is normal (just the amount of protein changes) b/c has no effect on transcription units, which code for RNA

^ basically functions of enhancers