Lecture 1 bio 2

nucleic acids

polymers specialized for storage, transmission, and expression of genetic info

• monomer= nucleotide (composed of pentose sugar, nitrogenous base, and phosphate group)

Pyrimidines

Cytosine & Thymine

Purines

Adenine & Guanine

DNA is…

double stranded, supercoiled and antiparallel

2 reasons protein was initially thought to be genetic material

more variety amongst proteins & more abundent and active in cell

Chargaff rules

amt of A = amt of T

amt of G = amt of C

Xray crystallography

method for determining 3D structure by analyzing how x-rays diffract through a crystalized molecule

what was Xray crystallography used for

discovering DNA’s structure

• DNA is helical

• DNA has a phosphate backbone

• 2 nm diameter, 3.4 nm long turns, 10 nucleotides in each turn

DNA basic structure

• sugar-phosphate backbone on outside

• nucleotide bases on inside

• complementary base pairing: A&T C&G

• double stranded, anti-parallel

DNA strands are held together by…

complementary base pairing with H-bons and van der waals forces

sugar-phosphate backbone provides…

stability and strength

Phosphodiester bonds are..

covalent bonds

number of H-bonds between C & G

3

number of H- bonds between A & T

2

H-bonds ensure…

DNA is always 2nm in width

vanderwaals ensure…

bases don’t collapse on each other b.c. the are between base stacked on each other

5’ end

phosphate group bound to 5’ carbon of deoxyribose

3’ end

hydroxyl group (OH) bound to 3’ carbon of deoxyribose

DNA is a ____ handed helix

right

outer edges of DNA

exposed in major and minor grooves which allows for different patterns for protein bonding; creates distinction

double helical structure of DNA is essential to fxn:

• storing genetic info via sequences of base pairs

• replication using complementary base pairing

• being susceptible to mutations allows permanent changes to encoded info

• coded info can be expressed as phenotype

DNA replication uses _______ replication

semiconservative

Initiation of DNA replication overview

double helix unwound by helicase

separates strands stabilized by single-stranded binding proteins

each strand is primed w/ RNA primers

Elongation: DNA replication overview

addition of complementary nucleotides (dNTPs) in 5’-3’ direction

catalyzed by DNA polymerase

Termination of DNA replication

occurs when all DNA has been replicated

RNA primers are replaced w/ DNA

DNA fragments are connected by ligase

pre-replication

preparation for DNA replication that starts w/ binding of pre-replication complex at ori sequence

pre-replication complex

a group of several proteins, including DNA polymerase

origin of replication (ori)

DNA sequence where helicase starts unwinding DNA & replication begins (in both directions)

Prokaryotic chromosomes usually have ___ ori sequence

1

Eukaryotic chromosomes have ____ ori

multiple

DNA helicase

unwinds DNA at ori

single stranded binding proteins

• keep DNA strand separated (DNA naturally wants to snap back together)

• provides stability and strength to single strand

replication starts at the ____ end of the template strand

3’

Primase

synthesizes an RNA primer

RNA primer

short “starter” strand of RNA

DNA polymerase

starts replication on 3’ end by adding new nucleotides to synthesize DNA until new DNA section is complete

dNTPs

used by DNA polymerase uses it to add new nucleotides to growing daughter strand to form phosphodiester bonds

DNA polymerase uses…

breaking of phosphate bonds for energy to attach the dNTP via phosphodiester bonds

Replication fork

opens like a zipper in one direction

leading strand

can be replicated continuously at its end as the fork continues to open

DNA replication occurs in the….

5’-3’ direction

lagging strand

oriented so as DNA unwinds, its exposed end gets farther away from the fork, and unreplicated gap is formed

okazaki fragments

new, short segments of DNA that make up the lagging strand in DNA replication

lagging strand is produced in…

short fragments w/ multiple primers and enzymes

leading strand grows…

continuously “forward” in 5’-3’ direction

lagging strand grows…

“backwards” discontinuously in 5’-3’ direction

different DNA polymerase….

replaces RNA primer w/ DNA, leaving a gap in sequence

DNA ligase

catalyzes phosphodiester bonds between adjacent Okazaki fragments, linking them & making lagging strand whole

Termination

occurs when all DNA has been replicated, RNA primers are replaced w/ DNA, and DNA fragments are connected by DNA ligase

efficiency of replication is achieved by

1. DNA polymerase is a processive enzyme: it doesn’t stop after adding 1 nucleotide

2. sliding DNA clamp- stabilizes, lubricates, and ensures tight binding

3. replication complex remains stationary while DNA moves through it

problem w/ DNA replication

eukaryotic chromosomes loose part of their DNA w/ each cell division (50-200 basepairs)

telomeres

solve problem w/ replication; highly repeated sequences at end of linear chromosomes in many eukaryotes

• these segments of DNA are meaningless, so losing them doesn’t matter

telomerase

some cells use telomerase to avoid losing telomeres

• contains RNA sequence used as template & adds missing parts of telomeres

• expressed in stem and germ cells

PCR requires

1. sample DNA template

2. 2 synthesized DNA primers (complementary to each end of template sequence of interest)

3. the 4 dNTPs (dATP, dTTP, dCTP, dGTP)

4. taq polymerase - can withstand extreme temps.

5. buffer

6. specific thermal conditions (to unwind DNA, bind primers, & for polymerase fxn)

PCR uses ___ to split

heat

4 stages of PCR

1. denaturing

2. annealing

3. extension

4. repeat

mutation

permanent changes in the nucleotide sequence of DNA

mutations can be created ____ & _____

internally- when DNA polymerase makes a mistake

externally- chemicals, radiation, some viruses

3 methods of DNA repair

1. proofreading

2. mismatch repair

3. excision repair

DNA proofreading

corrects base pair mismatches as DNA polymerase makes mistakes

• immediately excises incorrect nucleotide and adds correct 1

Mismatch repair

Performed by proteins by scanning new strand immediately after replication & correcting mistakes

mismatch repair proteins identify which strand was replicated by…

1. looking at the cuts that haven’t been sealed by ligase in the daughter strand (since this process is immediate)

2. parent strand has all molecular additions

3. consistent diameter in normal DNA so kink can be identified (less important)

4. directionality (less important)

Excision repair

removes damaged based & replaces them w/ functional bases during life of cell (caused by external factors)

excision repair mechanism can identify mutations by…

looking a diameter of DNA (consistent in regular DNA)