Biomg 3300-Unit 6 Quiz

Adenine

Guanine

Cytosine

Thymine

uracil

Draw the structure of 2'-deoxynucleoside

some notes: side is only the sugar and the base, therefore it is CH2OH

deoxy= 2' carbon has an H instead of OH

note: there is an OH where the purine and puridine usually add just don't see it

they add where the NH is

Draw 2'-deoxynucleotide

with the phosphate

Draw 2'-deoxynucleoside 5'-di- or triphosphate

ok

Draw the numbering convension for pentose

ok

What atom in the sugar is the base attached to? What is this bond called?

1

N-B-glycosyl bond

Identify atom found in 2' position in DNA vs RNA

DNA: H

RNA: oh

What is the difference in the bases between RNA and DNA?

RNA: uracil

DNA: thymine

Describe how nucleotide polyermization occurs, draw it out?

the 3' OH attacks at the phosphate group (need triphosphate)

this allows DNA to run in 5' to 3' direction, shows that it can only be polermized in this direction

starts with phosphate and ends with sugar

the phosphate is partially pos

forms phosphodiester bond

LeChat: by removing a product, we drive the rxn forward. We are breaking the pyrophosphate into 2 phosphates which drives the rxn forward

Where is the phosphodiester linkage?

in the phosphate in the middle

What is the overall charge?

-

Distinguish between oligonucleotide and polynucleotide?

olig: short nucleic acid, polymer containing fewer than 50 nucleotides

polynucleotide: longer nucleic acid

Describe the H bonds?

A and T= 2 H bonds

C and G= 3 H bonds (higher MP, harder to pull)

Discuss the stucture of double stranded DNA?

major and minor grooves (ask)

the sugar phosphate backbone is polar so it is on the outside

hydrogen bonds between base pairs

bases are aromatic so they are nonpolar --> pushed to the inner of the helix

What 2 forces stabilize the DNA double helix?

metal cations: shield the negative charges of the backbone of phosphates (In DNA poly: has Asp residues which stabilizes Mg which stabilizes the DNA)

base stacking interactions between successive base pairs

What is the idea of base stacking? Where are the bases located relative to the polar backbone?

nonpolar pushed inside

the bases are perpendicular to the backbone

What bonds are broken during denaturation?

disruption of H bonds between paired bases and base-stacking interactions

no covalent bonds are broken during denaturation

Describe experimental procedure for generation denaturation graph? How is % denaturation quantified?

subjected to extremes of pH or temp above (80)

- detected by monitoring UV absorption at 260 nm

% denaturation: A260-Anative/(A den- Anative)

What is the MP tm?

Temp at which 1/2 the DNA is presented as separated single strands

What is the relationship between tm and base comp?

higher the content of C-G pairs, higher MP

What is annealing?

when unwound segments of the 2 strands spon rewind

hydrogen bonds are the ones that reform!

What is a gene?

All of the DNA that encodes the primary sequence of some final gene product which can be a polypeptide or RNA with structural or catalytic function

What distinguishes a plasmid from a chromosome?

Plasmids are small, circular DNA molecules free in cytosol, carries genetic information and undergoes replication for daughter plasmids (bacteria, yeast, fungi), nonessential gene info

Chromosome: linear, contains genetic info for organism that is inherited. Found in both pro and eu.

Is all eukaryotic DNA stored in the nucleus?

No, some eukaryotic DNA is found in mitochondria and chloroplasts.

Intron

nontranslated DNA segments

exon

coding segment of DNA

satellite DNA

highly repeated, nontranslated segments of DNA in eukaryotic chromosomes, most often associated with centromeric area

Centromere

specalized site in chromosome

serves as attachment point for mitotic or meiotic spindle

Telomere

specialized nucleic stucture at ends of linear chromosome

helps stabilize the chromosome

protective caps of rep DNA

What is meant by linking number?

number of times closed circular DNA is wound abt another

also could be number of loops

How do you calculate linking number of DNA molecule in fully relaxed state?

BP/10.5

What is a topoisomer?

different forms of covalently closed circular DNA molecules that ONLY differ in linking number

When does linking number change?

only when you break the DNA

How does over-winding or underwinding the DNA double helix lead to pos or neg supercoils?

By increasing or decreasing the linking number

overwinding: DNA double helix is more twisted tightly than its relaxed state. Coils on itself. Forms pos supercoil. Right handed. increasing linking number

underwinding: DNA has fewer twists than its relaxed state. Favors partial unwinding. DNA coils in opp. direction of helix, promoting - supercoiling. decreases linking number

Why is it advantageous for cells to maintain DNA in underwound state?

1) allows for packaging of DNA (either)

2) underwinding facilitates strand separation making it easier for enzymes involved in DNA replication and transcripton come in

What do topoisomerases do?

enzymes that introduce pos or neg supercoils in DNA

What is the difference between type 1 and type 2 topoisomerase?

type 1: breaks one of the 2 DNA strands, passes the unbroken strand through the break, rejoins the broken ends, changes Lk in incraments of plus/minus 1

type 2: breaks both DNA strands and changes in Lk of incraments of plus/minus 2

Describe in specific how Type 1 changes?

1) Active site Tyr attacks a phosphodiester bond in one DNA strand, cleaving it and creating a covalent 5'-phosphotyrosyl protein DNA linkage

2) Enzyme changes to open conformation

3) The unbroken DNA strand passes through the break in the first strand

4) Enzyme in closed conformation. Liberated 3'-OH attacks the 5'-hopsphotrysol protein DNA linkage to religate the cleaved DNA strand

How is DNA packaged into nucleosomes?

histone-octomer structure

beads on string

8 histone: 2 copis each of H2A, H2B, H3, H4

200 bp, 146 bound around histone, rest part of linking

What is unique abt the AA comp of histone proteins?

made of AA residues such as arginine and lysine (need basic + residues so it can attach to DNA)

What is meant by semi-conservative replication?

Each DNA strand serves as a template for the synthesis of a new strand, producing 2 new DNA molecules each with one old strand and one new strand

What is the origin of replication?

the nucleotide sequence in DNA where replication is initiated

What is the replication fork? What is bi-directional replication?

the y shaped structure found where DNA is synthesized

bidirectional: both ends of loops have active replication forks

Disntguish between a exonuclease and endonuclease

exo: degrades nucleic acids from 1 end of molecule, in 5 prime to 3 prime, or 3 prime to 5 prime

endo: degrades at specific internal sites in a nucleic acid strand or molecule

What are the 2 central requirements for DNA polymerase?

needs a template and a primer: strand segment complementary to the template with a free 3' hydroxyl group to which the nucleotide can add

Which type of nuclotide can be used as a substrate? Draw out arrow pushing? What drives the rxn forward?

need dNTP

draw out

by removing the 2 phosphates (which are in the product), the rxn is driven forward

DNA (n) + dNTP --> DNA (n+1) + PPi (pyrophosphate)

pyrophosphatase breaks down pyrophosphate which then pulls the rxn forward

Define processivity

average number of nucleotides added before a polymerase dissociates

How does the geometry of the active site of DNA polymerase contribute to the fidelity of DNA rep?

The standard AT and GC base pairs have similar geo and an active site used to fit one will generally accomodate the other

the geo of incorrectly base pairs can exclude them from the active site on DNA polymerase

Discuss proofreading by DNA polymerase? Which exonuclease activity is responsible for this?

3' to 5' exonuclease activity

polymerase will reposition the mispaired 3' terminus into the 3'--> 5' exonuclease site and exonuclease will hydrolyze the mispaired base

3' terminus will go back into the polymerase site

polymerase will incorporate the correct nucleotide

Compare DNA pol 1 and 3

3 to 5 exonuclease

5 to 3

rates of polymerization

processivity

1: yes, yes, 10-20, 3-200

3: yes, no, 250-1000, greater than 500,000

Why is DNA pol 3 more processive than DNA pol 1?

due to addition of B subunits

the b subunits associate in pairs to form donut shaped structures that encircle the DNA and act like clamps

b sliding clamp prevents the DNA pol 3 from disassociating from the DNA which increases processivity

What overall does topoisomerase do?

strand separation creates topological stress in the DNA structure which is relieved by topoisomerase

What do DNA binding proteins do?

stabilize separated strands

What does primase do?

synthesizes RNA primer to allow DNA polymerase to begin replication

What 2 things does DNA ligase do?

After RNA primer is removed and filled with DNA

some nicks are in the DNA which ligase helps fill and it seals the okazaki fragments together

Discuss the sequence features at OriC?

origin of rep in prokaryotes

DUE repeats: 3 13 bp sequences, rich in AT h bonds so they can be easily denatured

R sites (repeat of 5 with 9 bp sequences)

I: 3 repeats

Both R and I bind DnaA which binds and hydrolyzes ATP

R: binds DnaA in the active or not active site

I: binds DnaA only in the active site (bound to ATP)

allows for discrimiation between active and inactive forms of DnaA

8 DnaA molecules bind across R and I--> also binds to each other -> this creates supercoiling and tension which breaks open DUE and DUE is denatured

we want the active form bc this means we have enough energy to do this

What is the helicase and the role of it that comes in next?

DnaB=helicase

migrates along and unwinds the DNA as it travels

What is gyrase?

a type 2 topoisomerase that acts upstream of the DNA that introduces negative supercoils to counteract the pos supercoils creates outside of the replication bubble (reduces linking number)

as DNA is unwinding, pos supercoils are added ahead of the replication fork

What protein binds to the resulting single stranded DNA? Why is this important?

SSB: keeps single strand from renaturing into something else

What process is required before DNA synthesis can occur on the single stranded template? What enzyme carries this out? Why is RNA used? What enzyme then carries out the bulk of DNA rep?

short RNA primer needs to be synthesized by primase (dnaG protein)

DNA polymerase requires an existing 3' OH group to initiate synthesis

DNA poly 3

Discuss how a dimer of DNA pol at the replication fork can copy both parental strands at the same time?

Lagging strand is looped

Has 2 core proteins with clamps on both strands

in the middle has a clamp loading complex with an open B sliding clamp

Okazaki fragment nears completion and primase binds to DnaB (helicase) to synthesize a primer and disassociates

A new B clamp is loaded by the loader and synthesis of the okazaki fragment is done

lagging strand core subunit are transferred to the new template primer and its B clamp, the old B clamp is leftbehind

the next B clamp is readied as Okazaki fragment synthesis is initiated

Name the DNA pol that removes the RNA primer, what activity is required?

DNA poly I

5' to 3' exonuclease activity

DNA poly I replaces RNA with DNA

the remaining nick is sealed with ligase (nick on right)

Name the enzyme that catalyzes the formation of phosphodiester bonds between the newly synthesized DNA fragments. Name the source of energy?

ligase

ATP

needs ATP bc normally nucleotide polymermization comes from the pyrophosphate

Discuss the role of Ter sequences in termination?

Ter: 20 bp sequence bound to Tus

can arrest a replication fork in only 1 direction

1 ter complex per rep cycle

opposing rep forks generally halt when they collide, but this may prevent overreplication by 1 fork in the event that the other is delayed due to mutation

fork meets Ter --> halts, the other fork halts when it meet arrested fork

What does this create? What separates them?

2 topologically interlinked (catenated) circular chromosomes --> catenanes (happens in ecoli, need to separate for daughter chromosomes)

needs topoisomerase iV (type 2 isomerase)

Compare the number of origins of rep in E coli to the number of eu?

E coli: single origin

Eukaryotes: many origins per chromosome

What are telomeres?

DNA at the tips of chromosomes (repeated sequences), not easily replicated, protective cap (in eu due to being in linear chrom)

Why can't linear chromosomes be copied by lagging strand synthesis?

there is not enough DNA to put an RNA primer to make an okasaki fragment so a short part of the DNA does not get replicated

Name the type of enzyme that adds telomeric ends? What type of enzyme is it?

telomerase: reverse transcriptace bc it used RNA to make DNA

Discuss telomere addition? What serves as the primer? What serves as the template?

3' end of DNA is primer

telomerase has its own internal RNA template (CA) which then results in TG strand synthesis

this extends the template strand

telomerase disassociates, RNA primase makes RNA primer at end of telomere strand

DNA poly fills in intervening gap

DNA ligase seals nick

RNA primer removes RNAase

single stranded part of the telomere end is protected by telomere binding proteins

GERMLINE HAS TELOMERASE

Discuss the role of telomeres in cell senescence?

In somatic cells, telomeres are not lengthened, and as telomeres shorten, individual ages (leads to cell senescence-cessation of cell divisio

List 3 key processes that contribute to the high fidelity of DNA rep?

1) accurate base selection by DNA poly (incorrect bases do not fit into DNA poly)

2) proofreading by 3'-5' exonuclease activity

3) post replicative mismatch pair

How is methylation used to distinguish the parental strand in DNA rep from the newly synthesized strand?

mismatch repair

enzyme dam methylase methylates all A's at the N^6 position of all adenines with the 5' GATC sequences. There is a short period where the template strand is methylated and the newly synthesized strand is not. This allows them to be distinguished

Discuss how cells distinguish which nucleotide is correct when a mismatch is detected?

MutS scans the DNA and forms a clamplike complex when seeing a leison (complex then binds to all mismatched pairs, except C-C)

Mut L forms complex with MutS and MutSL slides along complex to find hemimethylated DNA

Mut H then binds to Mut L and the complex moves in either direction at random along the DNA. MutH has a site specific endonuclease activity that is inactive until seeing hemimethylated GATC sequence

Mut H catalyzes the cleavage of the unmethylated strand on the 5' side of the G in GATC which marks strand for repair

Describe how the incorrect nucleotide is removed and replaced?

unmethylated strand is unwound and degraded in the 3'-5' direction from the cleavage site and replaced with new DNA

Discuss the spon cytosine deamination rxn?

cytosine turns into uracil (loss of amine group)

This is why DNA contains thymie instead of uracil, if this happened, it would not be able to detect it

Discuss base-excision repair, naming each enzyme and discussing the function of each?

DNA glycosylase: recognizes the leison and cleaves the N-glycosyl bond

AP endonuclease: cleaves phosphodiester bond

DNA pol 1: in 5 to 3 exonuclease activity, this replaces the DNA that was removed

DNA ligase: seals remaining nick

Does RNA contain equal amounts of guanine and cytosine?

No, it is single stranded

What type of bond is the N-glycosidic bond?

beta

Does RNA poly need a primer?

No

What are the 2 things DNA poly 1 does?

removes RNA primer and is used in DNA repair model

What does helicase look like and what does SSB look like?

helicase: one hold

SSB: multiple

Discontinuation replication is the result of which feature in DNA?

antiparallel

What are the 2 steps immediately after helicase unwounds the DNA?

SSB are added (need to prevent reannealing)

Then RNA primers are added

Then DNA poly 3

Then primers removed from DNA 1 and replaced with DNA

then ligase

What do some cancer drugs target?

topoisomerases: if these are targeted, then DNA replication cannot occur (need the underwound state in order to have efficient strand separation)

Explain how DNA poly 1 removes RNA primers? Do the rxns?

replaces NMP (primer) with dNTP

- rnaASE can also do this

Does ligase also use pyrophosphate?

yes, also uses this phenomenon to do reversible rxn