Ch 13: Recombination DNA repair and homologous recombination

RecA recombinase

enzyme for double stranded break repair

DNA photolyase (not in humans)

enzyme for photoreactivation (pyrimidine dimers caused by UV light)

DNA glycosylases

enzyme for base excision repair (oxidative damage; alkylated bases)

MutS and MutL

enzymes for mismatch repair (mismatched bases by polymerase)

excinuclease

enzyme for nucleotide excision repair (bulky alkylated bases; pyrimidine dimers)

TLS Pol

enzyme for translesion synthesis (abasic sites; alkylated bases; pyrimidine dimers)

other strand (can’t be used for double stranded breaks)

what is used as template in base excision repair (BER) and nucleotide excision repair (NER)?

nucleases and helicases

• degrades 5’ → 3’ in both strands to form 3’ overhangs (free 3’ OH)

• first step of double strand break repair

D-loop

• formed when 3’ overhang “invades” and pairs with DNA on homologous strand

• second step of double strand break repair

recombinase

• forming the D loop (1st 3’ overhang pairs with homologous strand) requires which enzyme?

• second step of double strand break repair

other homologous strand

• second strand with 3’ overhang invades ______

• third step in double strand break repair

DNA polymerase I

• extends 3’ OH using homologous strand as template

• fourth step of double strand break repair

RecBCD

complex with helicase and nuclease that binds to end of dsDNA and produces ssDNA with 3’ overhang

RecD

• helicase that unwinds dsDNA 5’ → 3’

• part of RecBCD

RecB

• helicase that unwinds 3’ → 5’

• has nuclease domain that degrades DNA

• part of RecBCD

RecC

recognizes chi site (5’-GCTGGTGG-3’)

chi site

when RecBCD reaches _____, it DECREASES degradation of 3’ end and INCREASES degradation of 5’ end

RecA

loaded onto 3’ overhang

single strand binding protein (SSB)

nucleation to form 3’ overhang is slow because ssDNA is bound to ____

RecB

• recruits the first RecA

• rest of the RecA rapidly binds cooperatively (binding of each RecA increases binding of additional RecA)

RecA

coats the ssDNA forming a ____ “filament”

RecA

• DNA-dependent ATPase

• hydrolyzes ATP → ADP when bound to DNA to drive homology searching and pairing

synthesis-dependent strand annealing (SDSA)

• option one after DNA pol extends strand in Holiday junction

• helicase melts complementary regions so that the strands dissociate from homologous strands

• DNA pol and DNA ligase complete replication and ligate

Holiday junction

four-branched crossover when both strands of broken DNA are bound to the homologous strands

branch migration

branch points in Holiday junction can slide back and forth at no energetic cost

non-crossover products

• resolution of Holiday junction where both junctions are cut the same way (X x X or Y x Y)

• X x X is where both outer strands are cut on both sides

• Y x Y is where both inner strands are cut on both sides

• no recombination

crossover products

• resolution of Holiday junction where each junction is cut differently (X x Y or Y x X)

• X x Y is where the outer strands are cut on the left side and the inner strands are cut on the right side

• Y x X is vice versa

• has recombination but the strands are identical so can’t detect difference

RuvAB (Repair of UV damage)

enzyme complex that stimulates branch migration and resolution of Holiday junctions

RuvA

recognizes Holiday junction and forms a complex with 2 RuvB hexamers

RuvB

• ATPase that uses energy of ATP hydrolysis to drive branch migration

• part of RuvAB complex

RuvC nuclease

• resolves Holiday junction

• recruited to RuvAB-Holiday junction and cleaves DNA

RecG

helicase that promotes fork regression

RecFOR complex

binds DNA and loads RecA onto ssDNA gap

SOS (DNA repair) gene

DNA-bound RecA induces ____

LexA

• constantly cycles on and off its binding site

• an abundant amount of this molecule ensures SOS genes are repressed because there’s always one bound to the gene

LexA

RecA bound to ssDNA induces SOS gene by inducing free ___ to cleave itself → SOS gene expressed

double strand break repair

• key aspect of recombination during meiosis 1

• creates genetic diversity and allows separation of favorable/unfavorable alleles

double strand break (DSB)

homologous recombination begins with ___

Spo11

makes a double strand cut for homologous recombination

Spo11-DNA

removed by Mre11-Rad50-Xrs2 as part of homologous recombination (part of DNA around the break is removed too leaving a 3’ overhang)

Sae2 and Sgs1

remove more DNA from 5’ end at DSB in homologous recombination leaving 3’ overhang

RecA

protein that is loaded onto 3’ overhang in homologous recombination to start strand invasion and pair with it’s homologous complement

resolvases

cleaves double Holiday intermediate in homologous recombination

recombination frequency

1 centimorgan (cM) = 1% _______ (1% chance that crossover will separate them)

hotspot

region of DNA where recombination occurs at higher frequency

30,000 (every 50-100 kb)

approximately how many hotspots are there in humans?

100

frequency of recombination (map units) can differ ___-fold within a given length of DNA (physical distance in bp)

recombination frequency

nucleosome density, histone variants, euchromatin vs. heterochromatin, and chromosomal loops are correlated with the difference in ______ given a length of DNA

recombination (crossover)

non-sister (homologous) chromatids don’t pair during mitosis so they don’t undergo ______ (can occur at low frequency)

somatic (mitotic) recombination

recombination occurring between non-sister (homologous) chromatids at low frequency during mitosis

cancer cells

somatic (mitotic) recombination occurs more often in ____ (progressive loss of growth control as they “evolve”)

sister chromatids

DSB repair using _____ should result in identical chromatids so no gene conversion

non-sister chromatids

• DSB repair using _______ results in allelic differences so there is gene conversion

• mismatches between the non-sisters → “repair” to fix the mismatch → gene conversion

translesion synthesis

allows DNA replication to continue by bypassing the lesion in template strand (lesion would otherwise block the replication fork)

lagging

translesion synthesis (bypass lesion during replication) is more frequent on ____ strand

fork regression

• when DNA pol encounters lesion in leading strand, daughter strands zipper back together over lesion (forms Holiday structure)

• gives it time to repair lesion (MMR, BER, or NER) → replication restarts by pushing inner strands into branch to unwind rep fork

exogenous DNA (Cas9 gene produces Cas9 protein)

DNA from outside the cell used to repair double strand breaks by homologous recombination

Cas9 protein

introduces double strand break in CRISPR gene editing

homologous recombination

introduce homologous donor DNA after Cas9 introduces DSB and it will be repaired by ____

CRISPR/Cas9

leaves no “fingerprints” so difficult to detect or block

CCR5

• coreceptor for HIV (if this gene is inactivated, you’ll be resistant to HIV)

• Chinese scientists used CRISPR-cas9 to inactivate this gene in babies

nonhomologous end joining

• last resort to repair DSB if all else fails

• mutagenic (induces mutations)

stress (antibiotics)

increases rate of mutation and helps cells develop resistance (adaptive evolution)

error prone DNA polymerase

• SOS response due to a DNA break leads to ____

• drugs that reduce induction of _____ → reduce formation of cells that are resistant to antibiotics

cipro

widely used antibiotic that inhibits gyrase (enzyme that introduces negative supercoiling)