9/13 Lecture 8: Double-stranded Break Repair and Recombination

Three types of transposons

1. DNA transposons -made of DNA 2. Retroviruses and retrotransposons -made of RNA, forming an intermediate in the transposition mechanism -ex: HIV, herpesvirus 3. Poly-A retrotransposons -made of RNA, forming intermediates in the transposition mechanism -LINEs and SINEs

HR mediated repair mechanism (Classical/meiotic mechanism)

1. Initiated by DSB on one chromatid/chromosome -DBSs formed by DNA damaging agents can have abnormal ends -Meiosis: programmed DBS are generated by Sop11, a meiosis-specific nuclease 2. Resection of breaks to form long 3' overhangs -5'->3' exonucleolytic processing (+/- abnormal end processing) -mammal cells require MRN complex plus CtlP 3. Filament formation on single stranded 3' overhang -RecA (E. coli), Rad51 homologs (eukaryotes) -meiosis: Rad51 and its homolog DMC1 are required -Rad51 creates the 1 strand complex with the 3' overhang 4. Strand invasion of filament into homologous sequence and strand exchange, resulting in displacement(D)-loop formation -3 stand complex of the duplex, the 3' overhang, and Rad51 5. a) DNA synthesis and b) second strand invasion/pairing -with identical duplexes, as for sister chromatids, DNA synthesis restores sequences "lost" at break and during resection -if duplexes are not identical, as for different alleles on homologous chromosomes, sequences from intact duplex are transferred to broken duplex causing a change in sequences 6. Branch migration and double Holiday junction formation 7. Holliday junction resolution by resolves -Mitosis: sister chromatid exchange at cross over is not usually observed (suggesting tight control at this stage), minimizing genetic change observed -Meiosis: crossing over occurs often between sister chromatids and is beneficial for genetic diversity -this indicates that resolution is different among mitotic and meiotic processes

Synthesis-dependant strand annealing (SDSA) HR model

1. Initiated by DSB on one chromatid/chromosome -DBSs formed by DNA damaging agents can have abnormal ends -Meiosis: programmed DBS are generated by Sop11, a meiosis-specific nuclease 2. Resection of breaks to form long 3' overhangs -5'->3' exonucleolytic processing (+/- abnormal end processing) -mammal cells require MRN complex plus CtlP 3. Filament formation on single stranded 3' overhang -RecA (E. coli), Rad51 homologs (eukaryotes) -meiosis: Rad51 and its homolog DMC1 are required 4. Strand invasion of filament into homologous sequence and strand exchange, resulting in displacement(D)-loop formation 5. DNA synthesis 6. Strand displacement after synths past the break point 7. Strand annealing, synthesis of the non displaced strand, and ligation

Genome modifications created by CSSR

1. Insertions 2. Deletions 3. Inversions

Importance of homologous recombination

1. Meiosis and genetic diversity -exchanges segments of homologous chromosomes to rearrange chromosome and genes for future generations -if this does not happen, the same genes would always be linked together 2. Double stranded break repair 3. Resolution of replication blockage and replication restart -accomplished by unconventional HR pathways

Break induced replication HR pathway mechanism

1. Nick on template strand results in a one-ended DSB 2. resection, filament formation, strand invasion, and repair synthesis, similar to conventional HR pathways 3. HJ resolution 4. Fork restart with DSB repair

Key filament forming/homology search factors

1. RecA homologs (bacteria) 2. Rad51 homologs (eukaryotes) these wrap around the DNA and are able to accommodate 1-3 strands of DNA -strand invasion step is one strand -once the D-loop formed, Rad51 hold all three strands of the 3' overhang and the homolog duplex

Sources of homologous DNA duplex

1. Sister chromatids in late S, G2 phases of the cell cycle -used most often -thought to be error-free 2. Homologous chromosomes in diploid organisms -called intermolecular information donor -can have some sequence differences, which results in error-prone sequences 3. Homologous sequence elements (on different chromosome) -can be an Intra or Intermolecular information donor -can have some sequence difference, which results in error-prone sequence

Common mechanism for generating genomic changes

1. Transfer of single element between locations 2. Copying elements to a new location

NHEJ mechanism

1. Undamaged DNA 2. Double strand break occurs 3. End binding and bridging -binding: Ku protein -bridging: DNA-PK and phosphorylated Artimis proteins 4. Microhomology mediated alignment -feathers ends together at the break point 5. Processing, Gap-filling, and Ligation

Importance of transposons in higher order eukaryotes

>50% of the human genome is derived from transposable elements transposition is the primary mechanism for expansion of genomes in higher eukaryotes LINE and SINE (long and short interspersed nucleotide elements) are poly-A retrotransposons that compromise a large percentage of the human genome -some of these are autonomous but most are non-autonomous -Alu sequences are SINEs former transposable elements in our genome have degraded repeat and cannot transpose

Choosing the double strand repair pathway

NHEJ primarily acts on double stranded break in G1, while HR is actively restricted to S and G2 phases This pathway choice depends on how the ends of double strand breaks are processed CtlP protein is required for the end resection of breaks to yield long 3' overhangs that are needed for the filament formation and strand invasion steps of HR -this protein is upregulated only in the S and G2 phases, facilitating end resection that initiates repair by HR -this does not occur in G1, so NHEJ occurs without end resection

Strand break generating agents

Oxidative DNA damaging treatments -Ionizing radiation (X-ray, gamma rays) -Chemical agents (H2O2, bleomycin/neocarzinostatin) able to make both single strand and double strand breaks

Enzyme mediated, programmed/targeted double strand break formation

SpoII makes double stranded breaks to initiate meiotic recombination during mitosis immunoglobulin gene rearrangements (Ig class switching, V(D)J recombination) -make double strand breaks to scramble the genes to increase diversity -used to scramble Ig gene to make different classes and increase diversity

Cre recombination mechanism

Used to make genetic knockouts tyrosine recombinase CSSR mechanisms Cre-Lox system is used for genetic engineering particularly for making conditional gene knockouts 1. first strand cleavage 2. first strand exchange 3. second strand cleavage 4. second strand exchange

HR as a source of missing information

a double strand break that has potentially lost sequence information at the break site can be repaired with sequence provided by an intact homologous duplex

Collapse of a replication fork

a special circumstance of HR replication converts a single-strand break to a double strand break there is no compatible end and must be converted by an unconventional HR process break induced replication HR pathway: -a cell evolved HR pathway to deal with blockage and collapse of replication forks caused by DNA damage -recombination factors are nearly essential for completing replication not yet well defined many bulky lesions and inter strand crosslinks block replication and may cause replication fork remodeling and/or collapse (result in DSB)

Overview of double-stranded break repair pathways

accidental break occurs->loss of nucleotides due to degradation from ends two options from here: 1. NHEJ during G1: end processing ligation steps -region with altered sequence due to nucleotide loss or untemplated additions 2/ HR during S and G2: copying process involving homologous recombination -complete sequence restored by copying from sister chromatid/second chromosome

Holiday junction resolution

accomplished by symmetric resolvases: -RuvC or RusA (E. coli), Yen1 (yeast), Gen1 (mammals)

Homologous

any two DNA duplex that share extensive sequence similarity with each other

Autonomous transposons

carry functional genes for their own integration/transposition all contain transposes/integrase -RNA based transposons also contain reverse transcriptase (RT) (only retroviruses/retrotransposons/poly-A retrotransposons) -the reverse transcriptase synthesizes DNA from the RNA intermediates

Symmetric resolvases

catalyze coordinating incisions in 2 strands near the HJ that can be directly ligated without any DNA synthesis cut two strands in a holiday junction that can be swapped and relegated to form two duplexes

Microhomology mediate alignment

due to the abnormal ends group at the break point, the cell must find way to hold the strands together a complementary sequence farther down stream that allows for base pairing of the two strands so that there is a force holding the strands together -can be one or more than one base pair the extra, skipped over base pairs can be ligated or sequence may be repolymerized and inserted

Inversion

elements on same DNA molecule get replaced in inverted repeat orientation

Deletion

elements on the same DNA molecule in direct repeat orientation get separated

Insertions

elements on two different DNA molecules come together

DNA double strand breaks

extremely deleterious and cytotoxic both strands are impacted often have dirty 3' and 5' ends that cannot be simply ligated back together -not 3' OH and 5' PO4 groups -only adjacent 3' OH and 5' OO4 ends can be ligated -only 3' OH ends are appropriate for DNA synthesis repaired by two pathways: 1. Non-homologous end-joining (NHEJ) 2. Homologous recombination (HR)

Synthesis-dependant strand annealing (SDSA)

favored HR model in mitotic cells SDSA model is consistent with findings that most DSB repair by HR in mitotic cells occurs without crossing over uses homologous region on sister chromatid (or homologous chromosome) as template for strand invasion and DNA synthesis once synthesis has passed the original break site, this strand can be displaced and annealed to the other broken end to bridge the break -you then use the newly synthesized portion to synthesize the complementary strand -double HJ formation does not occur and HJ resolution is not needed, meaning crossing over does not occur

Filament formation, homologous pairing, and strand extrusion

filament formation is initiated on single stranded 3' overhangs formed by resection in the strand invasion step, these filaments bring in partner duplex for sequence homology search and confirmation confirmation of sufficient homolog triggers strand exchange and D-loop formation

Diploid organism

have pairs of homologous chromosomes, one derived from each parent, that are similar but not completely identical in organization and sequence along the entire length ex: different alleles of the same gene on homologous chromosomes would have some sequence differences

Activate ATM-dependent checkpoint pathway

includes p53 upregulation lies upstream of p53 important for genome stability and removing damaged cells often lost in cancer cells

Conservative site-specific recombination (CSSR)

involves movable DNA elements containing specific sequence regions (for recombinase binding and cleavage) that define the sites of integration, deletion, or inversion -requires 2 short regions of homology, each symmetrically surrounding a recombinase cleavage site certain viruses utilize integration/insertion (lysogenic phage) and excision/deletion (lytic phage) events as part of their life cycle all events require recombinases (AKA integrases)

Non-autonomous transposons

lost functional genes for its own movement movement depends on enzymes (transposases +/- RT) produced from intact autonomous transposons

Defects in genes involved with HR pathway

may increase cancer susceptibility ex: inherited BRCA1 or BRCA2 gene defects increase breast/ovarian cancer risks

Transposition

movement of defined genetic elements (transposons) into or within a genome mechanism for integration and excision of certain viral genomes target site is generally random, making it different from CSSR -can disrupt or alter their expression requires transposase/integrase encoded by a gene within a transposon -defined incision preserves elements and allows its repeated transfer or duplication three types of transposons 1. DNA transposons 2. Retroviruses or retrotransposons 3. Poly-A retrotransposons all DNA transposons and retrotransposons contain terminal inverted repeats at the ends of the elements all transposons are bordered by copies of the target site due to how these elements are transferred for copied

Other recombination pathways and role in genetic change

not used to repair DNA damage -create more genetic change more genomic or viral DNA elements into, out of, and between different sites on chromosomes -these pathways cause genetic changes and have substantial impacts on chromosome composition -have the potential to interrupt and alter gene regions and thus are an important mutation and evolutionary mechanism unlike HR, long regions of sequence homology are not needed, but short regions of homology (direct or indirect repeats) associated with the recombining elements are required mechanisms are very different from HR -utilize enzymes (recombinases/integrases/transposases) that both incise DNA backbone and exchange strands in a coordinated manner two pathways: 1. Conservative site-specific recombination 2. Transposition

Homologous recombination (HR)

requires use of intact homologous duplex DNA partner (sister chromatid) to repair the broken duplex used in replicating cells in S and G2 phases when the sister chromatid is available to serve as the identical homologous duplex partner early steps of HR between two homologous duplexes must include displacement/removal of a substantial stretch of the complementary strand of one duplex (resection), followed by identification and confirmation of sequence complementary between recombination partners (strand invasion and exchange)

Non-homologous end joining (NHEJ)

splices together broke ends, sometimes resulting in nucleotide sequence changes (most often deletions) around the break site most utilized in G1 and differentiate, closed mitotic, nonreplicating cells required for V(D)J recombination process of immunoglobulin diversity -individuals with defects in NHEJ factors have profound immunodeficiency because they cannot make full immune system diveristy

Error-prone

subject to mutation generation due to possible incomplete sequence homology types: -homologous chromosomes in diploid organisms -homologous sequence elements (on the same or different chromosome)

D-loop

the loop formed during strand invasion prior to holiday junction formation

Gene conversion using MMR

the same gene regions on homologous chromosomes often are 2 different alleles with some sequence differences (SNPs, deletions, insertions) after completion of meiotic recombination events between homologous chromosomes, nucleotide differences between invading strands and recipient strands result in heteroduplexes with mismatches or unpaired loops that are targeted by MMR -the MMR can target one strand or the other, which can result in a difference in alleles present on each of the homologs, resulting in difference seen in gamut cells correction of one strand by MMR converts this heteroduplex to one allele or the other, a process known as gene conversion

p53

transcription factor that regulates proteins causing cell cycle rest or apoptosis part of ATM-dependent checkpoint pathway