Ch17 DNA Replication, Repair, and Recombination

the cell cycle

G₀ → G₁ → S → G₂ → M (PMAT)

replication forks

formed where replication begins and proceeds bidirectionally, away from the origin

unwind DNA and copy both strands

theta replication

mechanism of replicating circular DNA

uses replication fork

replicons

replication site or replication bubbles

multiple per linear chromosome

origin of replication (and E. coli)

site of DNA replication initiation

OriC of E. coli is AT rich and about 245 bp in length

consensus sequences

common sequences conserved across various species

example is the AT rich oriC of E. coli

3 enzymes in E. coli that initiate replication

DnaA, DnaB, DnaC

each bind the oriC

role of DnaA (E. coli) in initiating replication

binds to the conserved sequence 9-mer of oriC

result is unwinding of DNA at 13-mer sites

role of DnaB (E. coli) in replication

acts as DNA helicase to unwind DNA strands

DNA replication initiation steps in E. coli

1) DnaA binds the 9-mer sequence

2) unwinding at 13-mer sequence

3) single stranded protein binds unwound regions

3) DnaB (helicase) unwinds DNA as sequence proceeds

4) DNA polymerase and more proteins are added

5) replication proceeds

DNA polymerase III vs DNA polymerase δ

III: replicative enzyme in E. coli

δ: replicative enzyme in eukaryotes

directionality of DNA replication

3’ (hydroxyl) → 5’ end

leading vs lagging strand

leading: synthesized continuously in 5’ → 3’ direction

laggind: synthesized in Okazaki fragments that make a 3’ → 5’ replicated strand

why do eukaryotes have telomeres

lagging strands are difficult to deal with because they need primers

each round of replication results in the loss of some nucleotides from the end of the sequence

repeated sequences at the ends of chromosomes solve this

composition of telomeres

110 - 1500 copies of TTAGGG

noncoding

telomerase

catalyzes addition of telomeres to chromosome ends

hayflick limit

amount of times a cell population can divide before reaching senesence (permenant cellular arrest) or apoptosis

how do cells circumvent the Hayflick limit

producing telomerase

link between telomerase and cancer

almost all cancer types have telomerases

telomere capping proteins

form T loop to protect linear ends of single stranded DNA

werner syndrome

patients lack a telomere cap protein WRN

premature aging

types of mutations that occur during DNA replication

spontaneous mispairing due to transient formation of tautomers (resonance structures of nitrogenous bases)

slippage (example is trinucleotide repeats)

spontaneous damage to individual bases (depurination and deamination)

most common mutation in DNA replication

mispairing due to tautomers

trinucleotide repeats

spontaneous replication error that occurs in a region with repetitive DNA

causes slippage errors

depurination DNA damage

loss of a purine

human cell can have 1000s/day

deaminations

loss of a bases amino group

human cell can have 100/day

Ethyl methansulfonate (EMS)

chemically alters base so it will mispair in next replication
adds an ethyl group to bases

nitroguanidine

chemically alters base so it will mispair in next replication

adds methyl groups

nitrous acid (HNO₂)

chemically alters base so it will mispair in next replication

increases likilihood of deamination

aflatoxin B1

add bulky DNA adducts to DNA

attaches to guanine and causes depurination

pyrimidine dimer

triggered by UV radiation

covalent bond forms between adjacent pyrimidines

ionizing radiation

removes electrons from molecules to generate damaging reactive intermediates

base excision repair and example

corrects single damaged bases

DNA glycosylase detects damage and cleaves base

steps of nucleotide excision repair

1) proteins detect distortions in DNA helix

2) recruits NER endonuclease to cut DNA on both sides of lesion

3) helicase unwinds DNA between nicks (incisions) and frees distorted sequence from DNA

4) polymerase and ligase finish repair

steps of mismatch repair

repairs abnormal nucleotides after DNA replication

1) MutS detects mismatch

2) endonuclease MutH introduces a nick in unmethylated strand

3) exonuclease removes incorrect nucleotides from nicked strand, and these are replaced with correct sequence

significance of methylation to mismatch repair systems

DNA methylation is not immediate after DNA replication allowing distinction between old and new strands

homologous recombination

uses the process of crossing over

homologue acts as template for accurate repair due to sequence similarity

purines vs pyrimidines

Purines: AG, two rings (AG is silver! you want more!)

Pyrimidines: CUT, one ring