Chapter 16 - Repair of DNA Double-Strand Breaks and Homologous Recombination

synthesis-dependent strand annealing (SDSA)

-strands in the repaired region are generated by DNA synthesis -no extended region of heteroduplex DNA formed between parental and newly synthesized strands -DNA synthesis begins in the D-loop -as synthesis proceeds, the newly synthesized strand is released from the template strand, and the D-loop translocates along the template strand -when it has synthesized past the site of the double-strand break, the newly synthesized strand pairs with the complemenary strand of the damaged duplex on the other side of the break -remaining single-strand gap is synthesized using the new DNA as a template

presynaptic step in bacteria (homology-directed repair)

-two presynaptic pathways: RecBCD (predominant) and RecF -mediated by protein complex called RecBCD -protein complex attaches at a double-strand break and translocates along the DNA, separating the two strands and cleaving the unwound strands into small fragments -Chi sequence: 8bp DNA sequence that modulates nuclease activity of the enzyme (RecBCD complex nuclease activity reduces when the complex arrives at a Chi sequence) -RecBCD complex degrades the 5' end strand leaving a duplex DNA with a single-stranded 3' tail containing the Chi sequence -Chi site stimulates repair because it promotes formation of single-stranded 3' end DNA

break-induced repair (BIR)

-uncaptured chromosome fragment can be replaced by continued DNA synthesis from the invading strand of the D-loop through BIR -capture of DNA extension is key to homologous recombination

gene conversion/loss of heterozygosity

-when a homologous chromosome provides the template for repair instead of a sister chromatid -does not restore original sequence

homology-directed repair steps

1. generation of single-stranded DNA at the site of DNA-strand break 2. pairing by one of the single-stranded ends with the intact duplex by invasion of the duplex to form a heteroduplex 3. repair of the damaged duplex by DNA synthesis using strands from the undamaged duplex as a template 4. the separation of the two duplexes -synapsis: formation of the heteroduplex

alternative lengthening of telomeres (ALT)

-ALT pathway depends on BIR in which the shortened ends can invade other chromosome end at similar sequences thus priming DNA replication that extends to the end of the template chromosome and adding a new telomere to the invading chromosome end

gene conversion

-DNA of a homologous chromosome serves as the repair template, the repair chromosome will acquire a new sequence - that of the homolog

generation fo ssDNA for strand invasion (eukaryotes)

-MRN: initiates repair process because its arrives early to the damaged site -MRX: MRN equivalent in yeast; associates with exonuclease Sae2 to start the resection process

RuvAB

-bacteria -RuvA is a tetramer that binds at the Holliday junction, keeping it in the unfoleded and open conformation -RuvA recruits RuvB to the junction -RuvB is a helicase that uses ATP to move the Holliday junction along the DNA, breaking and reforming base pairs in one direction -RuvC is a resolvase recruited to Holliday junctions by its interaction with the RuvAB branch migration machine

homology-directed repair

-can restore a collapsed replication fork caused by DNA damage -pg 645, figure 16.13

non-homologous end joining (NHEJ)

-double-streak break repair -the broken ends are simply rejoined -does no occur in E.coli -occurs predominantly in non-dividing cells -does not require a template, but can cause mutations (nucleotides at the breakage site are often lost though degradation by cellular nucleases before repair is initiated and further loss frequently happens because of trimming that occurs during the repair process)

directed gene conversion

-expression of a or alpha mating-type transcriptional regulators at a site called MAT on chromosome III -haploid cells can switch mating type by a process involving directed gene conversion by homology-directed repair -transcriptionally silenced copies of MATalpha and MATa at HML and HMR sites respectively -mating-type switching between alpha and a cell types occurs by the transfer of information from a silent HML or HMR locus to the MAT expression site (occurs by directed gene conversion)

step 1 of homology-directed repair steps

-generation of single-stranded 3/ ends at the double-strand break by specilaized helicases and exonucleases by degradation -requires long regions of homology at the break for base-pairing to occur -single-strand tails at the break are at least 50 bps long to ensure accurate pairing with the template DNA in another chromosome

Holliday junctions

-homologous recombination occurs by the formation and resolution of holliday junctions between homologous chromosomes -holliday junction is formed by the reciprocal exchange via D-loop formation by RecA-like recombinases mediated formation of DNa strands between two duplezes to form regions of heteroduplex DNA -if cleavage occurs in the horizontal direction, the duplexes are separated without exchange of the DNA outside of the holliday junction (non-recombinant) -if resolution occurs in the vertical direction, the DNA outside of the Holliday junction is exchanged or crossed over between the two parental duplexes (recombinant)

RecF pathway

-includes helicase RecQ and nuclease RecJ that generate single-stranded DNA -pathway used in the repair of replication forks that have stalled as a result of single-strand lesions in the DNA

homology-directed repair

-involves pairing between the damaged DNA and homologous sequences in other chromosome which serve as the template for repair -major mechanisms for repairing double-strand breaks that occur during or after DNA replication (S phase or G2 phase) -no mutation or loss of sequences

strand-exchange recombinases

-mediate reactions exchange the single strand with one of the strands of the duplex to form heteroduplex DNA -RecA proteins: bacteria -Rad51: eukaryotes -Dmc1: eukaryotes; collaborates with Rad51 to promote strand exchange during meiotic recombination

Ku

-necessary for NHEJ process -protein that binds to broken DNA ends and recruits the nucleases, polymerases, kinases, phosphatases, and ligases that mediate the joining reaction 1. Ku complexes slide onto each end of the double-strand DNA break and act as a scaffold for the recruitment of DNA repair enzymes, in particular a protein kinase subunit, DNA-PKcs, to form DNA-PK. 2. DNA-Pkcs in activated by binding to Ku, resulting in autophosphorylation, and also phosphorylates and controls other important repair proteins (Artemis - overhang trimming nuclease) 3. DNA end-processing enzymes process the ends before ligation 4. DNA ligase IV ligates the broken DNA ends

replication fork reversal

-pair the two newly synthesized strands of DNA with each other instead of with the template strands -fork reverses so that the damaged strand re-pairs with its complementary strand -strand re-pairing provides an opportunity for lesion repair by systems such as base and nucleotide excision repair which use the complementary DNA strand as a template -post repair: 1. regressed fork may be cleaved to yield a collapsed fork which may be repaired by homology-directed repair; 2. newly synthesized annealed strands may be degraded by nuclease; 3. the regression of the fork may be reversed `

presynaptic step (homology-directed repair)

-process of generation of the long 3' ended single-straded tails that are required for homology-directed repair -DNA helicases separate the two strands of DNA by unwinding -nucleases degrade one of the DNA strands at the broken DNA (at the 5' end)

RecA

-recombinase protein -loaded onto the single-stranded 3' end DNA generated at a Chi site by RecBCD -RecA promotes homologous pairing between the single-straded DNA and DNA duplex that will provide the template for repair

presynaptic filament

-strand exchange begins with the loading of the RecA monomers onto single-stranded DNA to form the presynaptic filament (requires ATP) -DNA binding promots the assembly of further RecA monomers -binding of RecA nucleoprotein filament to the DNA to the invaded intact duplex distorts the duplex DNA molecules so that stretches of extended and untwisted DNA alternate with 3-nucleotide segments that assume the conformation of B-DNA (promote correct base-pairing)

step 2 of homology -directed repair steps

-strand invasion: the single-stranded DNA from one end of the damaged duplex locates and pairs with its complementary strand in the related unbroken duplex to for a DNA heteroduplex -heteroduplex: a region in which a single strand from one DNA molecule is base-paired with a single strand from a different DNA molecule -forms a D-loop

holliday junction branch migration

-when two identical duplexes are joined at a holliday junction, the junction can move along the DAN without any net change in base-pairing -can occur in either direction over short distances -can increase or decrease the region of heteroduplex DNA between the linked duplexes

homologous recombination

1. D-loop between one of the broken duplexes and a homologous duplex 2. broken DNA ends are degraded to generate single-stranded 3' tails. These ends are bound by strand-exchange recombinase molecules, RecA or Rad51, or the specialized meiotic recombinase Dmc1 3. resulting nucleoprotein filament then invades the intact duplex 4. Dna synthesis with the invading 3' serving as a primer and its complementary strand in the participating homolgous duplex as a template 5. newly snthesized strand remains paired to the template in the homologous duplex and the D-loop is extended by continued DNA synthesis 6. gaps in the broken strands can be closed by repair polymerases using the free 3' OH end of the cut strands as a primer -semiconservative