Prac Quiz 5: Double Strand Breaks

What kinds of proteins are required for NHEJ?

- at least 9 different proteins are used in multiple steps of NHEJ - the basics: nucleases for making blunt ends, DNA polymerases for filling in ends, ligases for forming phosphodiester bonds - special: protein called Ku binds to broken ends and recruits nucleases, polymerases, kinases, phosphatases

What can DSBs cause?

CELL DEATH

What can occur as a result of NHEJ?

CHROMOSOMAL TRANSLOCATIONS: when two double-strand breaks occur on different chromosomes, rejoining is almost always done by NHEJ >>> can cause reciprocal translocations

What is step 1 for NHEJ?

Ku complex slides onto each end of DSB and acts as scaffold for recruitment of DNA repair enzymes >> NBS1, MRE11, RAD50

What is the primary repair pathway for DSBs in mammals and why?

NHEJ because mammalian cells are mostly in G1 and in G1, NHEJ is the only pathway available

How does NHEJ pathway contribute to the immune system?

NHEJ pathway provides the means for DNA joining steps in programmed rearrangement of DNA of specialized cells of immune system >> this rearrangement generates the highly variable proteins (ex: antibodies) that direct infection

Homology-directed repair SDSA and DSBR are similar but not identical processes. Which of the following steps are similar in the two processes? A. a 3' overhang invades the other DNA helix B. DNA ligase is required C. new DNA incorporated in both DNA helices E. two Holliday junctions are generated F. recombinase is required (many copies)

TRUE: A. a 3' overhang invades the other DNA helix B. DNA ligase is required FALSE: C. new DNA incorporated in both DNA helices >> this is only in DSBR, in SDSA, new DNA incorporated into one strand only E. two Holliday junctions are generated >> only DSBR F. recombinase is required (many copies) >> only DSBR

What is an example of a disease caused by reciprocal translocation in chromosomes?

chronic myeloid leukemia >>happens in a single bone marrow cell, results in clonal expansion how: BCR-Abl fusion gene and corresponding protein >> results in a fusion protein, chimeric protein >> ABL-tyrosine kinase is normally tightly regulated, but when fused to BCL it is not (fusion is called BCR-ABL1) >> this leads to uncontrolled growth = leukemia

What are the possible causes of double stranded breaks?

external sources: chemicals, reactive oxygen species, ionizing radiation biological sources: replication, nucleases, programmed breaks, etc.

After the two general steps that apply for all homology directed repair, what steps occur for DSBR specifically?

general: 1. resection 2. formation of heteroduplex specific to DSBR: 3. synthesis primed from invading strands 4. D-loop captured by other 3' overhang 5. synthesis primed from captured strand 6. result: two Holliday junctions

After the first two steps of general homology-directed repair, what are the steps to finish up SDSA (synthesis dependent strand annealing)?

general: 1. resection 2. heteroduplex formation specific to SDSA: 3. new DNA released from template, reanneals with damaged strand 4. polymerase fills in gaps 5. ligase seals ends

How are evolution and homology directed repair connected?

homology-directed repair provides the mechanism for the natural homologous recombination that occurs for.... 1. sex: meiotic crossing-over 2. immunity: creation of antibodies 3. DNA uptake: transformation These natural homologous recombinations contribute to genetic diversity and evolution

In this first few steps of homology-directed repair (shared by bth SDSA and DSBR)...how long are the tails usually made? How is the single-stranded tail generated? What is formed by the second step (strand invasion)?

how long: usually 50 nucleotides - long tails needed to ensure accurate pairing with template DNA in another chromosome and error free DNA replication repair how it's generated: by nucleases and helicases second step: forms heteroduplex >> displaces strand of the same polarity (the parallel strand) from the undamaged duplex with formation of a three stranded structure called displacement loop

What are some general characteristics of homology-directed repair of DSBs: how many errors, what type of template, when it occurs, what it requires?

how many errors: error-free repair process, no bases lost template: sister chromatid when: post-replication (after copying DNA) requires: long, single-stranded regions of DNA

Random insert question: Why is NHEJ so harmful to DNA?

in first step of resection (chewing back to get blunt ends) >> something important is deleted

What does the formation of the heteroduplex (in ALL homology-directed repair) require?

lots of proteins: 1. RecBCD complex binds to broken ends of DNA 2. RecBCD complex has helicase and exonuclease activities that prepare 3' single end 3. RecA binds and coats exposed single strand 4. locates, stretches homologous double stranded DNA 5. D-loop formation by strand invasion >> RecA proteins transfer to the top, looped and exposed DNA of the D-loop

Is the heteroduplex formed in homology-directed repair permanent?

no - transient in that the sequence that is replicated will eventually be released

What is the difference in how the D-loop is used in non-homologous repair versus homologous repair?

non-homologous: D loop translocates, moves homologous: D loop gets bigger

What is gene conversion? Describe mechanism.

one of the pathways for non-homologous end joining 1. DSB processed to produce 3' single-stranded tails 2. which recruit Rad51 and thereby seek out a homologous template on the sister chromatid or homologous chromosome from which to accurately resynthesizes the sequence surround DSB

What is the name of the enzymes that mediate ss pairing in homology-directed repair in creation of the heteroduplex? How do these differ between bacteria and eukaryotes?

recombinases bacteria: RecA eukaryotes: rad51

Heteroduplex

region in which strand from one DNA molecule is based paired with single strand from different molecule (formed in 2nd step of homology-directed double strand break repair)

What enzymes act on Holliday junctions in DSBR?

resolvases: 1. helicase unwinds dsDNA (branch migration) 2. endonuclease cleaves ssDNAs (resolution in two ways)

So in gene conversion, Rad51 can seek out a homologous template on the sister chromatid OR the homologous chromosome. Which is preferred and why? How is yeast different from this?

template from identical sister chromatid is preferred because it maintains genome integrity (using homologous chromosome as template would change it a bit) for some reason though, yeast uses homology-directed repair for most

When can gene conversions occur relative to a crossover or exchange event?

they can over in the absence (GC) or presence (CO) of a crossover or exchange event >> sister chromatid crossover events can be equal (error-free events termed sister chromatid exchange, SCE) or unequal depending on template used

Some microbes can withstand up to 200 DSBs without cell death - how?

they have multiple copies of their chromosomes - sometimes up to 15 copies - always provides a template for DSBR >> efficient repair mechanisms and multiple copies of repair proteins present at all times

NHEJ in general: when does it function? What is it? What does it often cause? What does it need and not need?

when: throughout cell cycle, mostly during G1 (it's the only pathway available in G1) what: joining broken ends by ligation causes: mutation because causes deletions and translocations does not require another DNA helix as template

Two types of homology directed repair of DSBs:

1. Synthesis dependent strand annealing (SDSA) 2. double strand break repair (DSBR)

What are the two main pathways with non-homologous repair?

1. gene conversion 2. single-strand annealing

What are the basic steps for homologous recombination during meiosis? How is this different from the mechanism used in DSB repair?

1. homologous chromosomes 2. chiasma (recombination) 3. resolution (crossing over) 4. recombinant chromosomes in DSB, repair happens (preferably) between sister chromatids >> but in meiosis, for the purpose of creating genetic diversity, the SAME mechanism for DSB repair is used but with HOMOLOGOUS chromosomes

What are the two main pathways of double-strand break repairing in DNA?

1. non-homologous end joining (NHEJ) - does NOT use template, imprecise 2. Homology-directed repair: template-dependent, precise, TWO main pathways

What steps do all homology-directed repair processes (SDSA and DSBR) have in common?

1. resection to expose single-stranded DNA 2. strand invasion: invasion of homologs duplex by one broken end to form D-loop

What are the two main pathways within homology directed repair?

1. synthesis-dependent strand annealing (SDSA) 2. double-strand break repair (DSBR)

In what two ways can the Holliday junctions be resolved?

1. vertical resolution 2. horizontal resolution creates two different combinations of chromosomes

How many Double Strand Breaks (DSBs) arise in dividing cells?

10 times per day

Double-strand DNA breaks, if not repaired, are incompatible with the ability of a cell to reproduce. This is not true of most other kinds of DNA damage. Why are double-strand breaks so much more consistently harmful if not repaired?

because double strand breaks mean you can lose or gain genes after cell division - if lose a part of a chromosome arm without a centromere, won't be segregated correctly, will just be floating around