DSB repair and next generation sequencing

Question 1

What are DNA double-strand (DS) breaks and why are they important?

Answer 1

Double-strand breaks (DSB) occur when the phosphodiester backbone of both strands of a DNA duplex break at the same, or nearly the same, place. DNA double-strand breaks threaten the integrity of the genome, and thus their efficient repair is critical to maintain genome stability. Loss of repair and subsequent genome instability leads to a wide variety of disorders.

Question 2

Examples of natural DSBs

Answer 2

Meiosis, Generation of immune diversity, both VDJ recombination and class switch recombination, DNA replication, when the replication fork encounters a nick in one template strand or stalls at fragile sites, or sites of topoisomerase failure, On insertion of retroviruses or retrotransposons into genomic DNA

Question 3

Examples of DSBs due to environmental damage

Answer 3

These include cosmic rays and radiation from soils. The damage from ionizing radiation can be both direct (deposition of energy) and indirect (clustered oxidative free radicals caused by the energy deposition in turn cause DNA breaks). Medical tests and treatments are another significant source of ionizing radiation. Many commonly used imaging techniques including X-rays and CT scans, as well as radiation treatment and some chemotherapeutic agents (e.g. bleomycin) cause DSBs.

Question 4

How are DS breaks sensed?

Answer 4

Two members of the phosphatidylinositol 3-kinase related kinases (PIKKs) family are principally used to sense DS breaks: 1) ataxia telangiectasia mutated (ATM) and 2) ataxia telangiectasia and RAD3-related (ATR). These are protein kinases used to signal DNA damage more generally than just double-strand breaks. The disease ataxia telangiectasia causes genetic instability, a predisposition to cancer, and neurodegeneration.

Question 5

phosphatidylinositol 3-kinase related kinases (PIKKs)

Answer 5

a family of Ser/Thr-protein kinases. Include ATM, ATR, and DNA-PKcs

Question 6

ataxia telangiectasia mutated (ATM)

Answer 6

has an important role in the response to ionizing radiation, by phosphorylating several key proteins such as p53, Mdm2, Chk1, Nbs1 and Brca1 in response to DNA damage. These phosphorylation events are, in part, responsible for the cell cycle arrest that is necessary for DSB repair. Cells in which ATM is mutated are defective in the arrest at both the G1 and G2 phases of the cell cycle. In addition, while normal cells exhibit a dose dependent inhibition of DNA synthesis following exposure to ionizing radiation, A-T cells display almost no alteration in their rates of replication.

Question 7

ataxia telangiectasia and RAD3-related (ATR)

Answer 7

a serine/threonine-specific protein kinase that is involved in sensing DNA damage and activating the DNA damage checkpoint, leading to cell cycle arrest.[3] ATR is activated in response to persistent single-stranded DNA, which is a common intermediate formed during DNA damage detection and repair.

Question 8

How are DS breaks repaired?

Answer 8

Two pathways are used: 1) DNA non-homologous end joining (NHEJ) and 2) homologous recombination (HR); multiple proteins are involved in both.

Question 9

Homologous Recombination (HR)

Answer 9

occurs during and shortly after DNA replication, in S and G2 phases of the cell cycle. It absolutely requires nearly identical DNA strands (homologous) of the unbroken sister chromatid. Using this perfect template, HR repairs the break perfectly, with no gain or loss of nt. Multiple steps are required to repair double-strand breaks by homologous recombination. First the broken ends are resected, i.e., processed to expose single-strands ending in 3’ OHs. The single-strands then invade the homologous DNA, using it as a template for synthesis to bridge the gap caused by the break. The crossover (Holliday junction) then has to be resolved back to two separate DNA double strands. Typically in DNA repair using the identical sister chromatid perfectly restores the break with no exchange.

Question 10

non-homologous end joining (NHEJ)

Answer 10

NHEJ proteins are required for resistance to ionizing radiation and for VDJ recombination and immunoglobulin class switching in the immune system. NHEJ simply brings together the broken ends, and ligates them, typically (but not necessarily) losing a few nucleotides (nt) in the process. Thus fidelity of this system is imperfect, but its advantages include quick repair at any time in the cell cycle, and the process is not sequence-dependent. Recall that the human genome has a lot of space (introns, intragenic regions) where the loss of a few nucleotides here and there is not likely to cause a problem. The somatic mutations left behind by imperfections in the NHEJ process accumulate over time; a typical somatic cell in a 70 year old human has ~2000 little “footprints” from this imperfect repair system. Unpredictable/imprecise repair by NHEJ contributes to antibody diversity

Question 11

Holliday junction

Answer 11

a junction between four strands of DNA. Different resolutions of Holliday junctions can lead to exchange of genetic information between the two chromosomes undergoing HR. This exchange is an essential feature of meiosis and can take two forms, gene conversion or crossing over. In some cases of misregulated homologous recombination, using the other chromosomal homolog as a template for double-strand break repair instead of the sister chromatid in somatic cells can lead to loss of heterozygosity (LOH) by these same mechanisms. Holliday junctions must be cleaved to produce recombinant molecules. Depending on the direction of cleavage, the resulting molecules will or will not have exchanged sequences flanking the region of heteroduplex DNA (crossing over).

Question 12

What determines whether HR or NHEJ is used to repair a given DS break?

Answer 12

Two tumor suppressors, BRCA1 and 53BP1, are pivotal regulators of the choice to repair a DS break using HR or NHEJ, respectively.

Question 13

BRCA1

Answer 13

The double-strand repair mechanism that BRCA1 participates in is homologous recombination, in which the repair proteins utilize homologous intact sequence from a sister chromatid, from a homologous chromosome, or from the same chromosome (depending on cell cycle phase) as a template. This DNA repair takes place with the DNA in the cell nucleus, wrapped around the histone. Several proteins, including BRCA1, arrive at the histone-DNA complex for this repair. one of the key protein targets of phosphorylation by activated ATM and ATR; phosphorylation recruits BRCA1 to a double-strand break. Proper control of normal BRCA1 activity is required for double-strand break repair by homologous recombination although the exact role(s) of this large (1863 amino acids) protein and its many interactions remain a topic of active investigation. Significantly, in BRCA1 mutant mice that exhibit embryonic lethality, tumorogenesis and chromosomal abnormalities, genetic instability can be phenotypically rescued by a 53BP1 deletion. Importantly, the genetic instability caused by mutations in the core HR machinery, e.g., BRCA2 or XRCC2, are not rescued by a 53BP1 deletion. These findings indicate that, in the absence of HR mediated by BRCA1, 53BP1 promotes deleterious NHEJ; thus, the proteins encoded by BRCA1 and 53BP1 compete to determine the correct DNA DS break repair pathway.

Question 14

53BP1

Answer 14

a large (1972 amino acids) protein that interacts with a plethora of other proteins. Recently, key findings indicate that 53BP1 plays a crucial role in DS break pathway choice, acting as a positive regulator of NHEJ by promoting the synapsis of distal broken ends and by antagonizing their resection.

Question 15

XRCC2

Answer 15

This gene encodes a member of the RecA/Rad51-related protein family that participates in homologous recombination to maintain chromosome stability and repair DNA damage. This gene is involved in the repair of DNA double-strand breaks by homologous recombination.

Question 16

What happens when DS breaks are mis-repaired or not repaired?

Answer 16

Mis-repair is the source of genetic instability with translocations being particularly bad. Loss of HR leads to gross genomic rearrangements and thus genome instability. Translocations most often occur when DNA DSBs are healed between different chromosomes by NHEJ. Failure to repair a DSB will lead to cell death, a fact exploited by cancer therapies (ie radiation and drugs that cause DSBs like bleomycin). DSB repair deficits, which increase cancer susceptibility, can be caused by loss of proteins involved in the signaling (both at the level of sensors and transducers) of the DSBs and in the enzymes that mediate their actual repair

Question 17

RAD51

Answer 17

plays a major role in homologous recombination of DNA during double strand break repair. In this process, an ATP dependent DNA strand exchange takes place in which a template strand invades base-paired strands of homologous DNA molecules. RAD51 is involved in the search for homology and strand pairing stages of the process.

Question 18

Next-generation sequencing

Answer 18

non-Sanger-based high-throughput DNA sequencing technologies. Millions or billions of DNA strands can be sequenced in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes. Direct sequencing of cDNA products. Can identify mutations and modifications. Direct measurement of splice form abundance. No limit on search space. Dynamic range ~ depth of sequencing. Need high coverage to quantitate low-abundance transcripts

Question 19

pacific biosciences

Answer 19

Uses SMRT (Single molecule real time) sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) – small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMRT technology developer, this methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases.

Question 20

illumina

Answer 20

In this method, DNA molecules and primers are first attached on a slide and amplified with polymerase so that local clonal DNA colonies, later coined “DNA clusters”, are formed. To determine the sequence, four types of reversible terminator bases (RT-bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides, then the dye, along with the terminal 3’ blocker, is chemically removed from the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.

Question 21

Issues that affect calling a SNP

Answer 21

Coverage (more coverage = more confidence), Error rates: library quality & sequencing platform-specific, Ploidy: need enough coverage to determine heterozygosity

Question 22

Exome sequencing

Answer 22

a technique for sequencing all the protein-coding genes in a genome. Goal: Build a sequencing library from non-contiguous parts of a genome. Sequence the ~1% of the human genome that codes for proteins. Used to identify novel coding SNPs among e.g. patients with same genetic disorder. The goal of this approach is to identify genetic variation that is responsible for both mendelian and common diseases such as Miller syndrome and Alzheimer’s disease without the high costs associated with whole-genome sequencing.

Question 23

Hybrid capture

Answer 23

Microarrays contain single-stranded oligonucleotides with sequences from the human genome to tile the region of interest fixed to the surface. Genomic DNA is sheared to form double-stranded fragments. The fragments undergo end-repair to produce blunt ends and adaptors with universal priming sequences are added. These fragments are hybridized to oligos on the microarray. Unhybridized fragments are washed away and the desired fragments are eluted. The fragments are then amplified using PCR

Question 24

Identifying chromosomal translocations using next-generation DNA sequencing

Answer 24

You can sequence lots of DNA, most of which doesn’t inform you about translocation OR you can sequence mRNA (~1% of the genomic sequence) and identify reads that come from different chromosomes Key idea: oncogenic gene fusions are expressed and provide a selective advantage (i.e. the locus is abundant in a population of cells)

Question 25

Non-invasive Whole-Genome Sequencing of a Human Fetus

Answer 25

13% of Mom’s circulating cell-free DNA is from baby. Inheritance of 2 million heterozygous sites predicted (98% accuracy). Not as effective for de novo mutations predicted. Light haplotyping and shallow sequencing can predict baby’s genome (but not de novo’s)

Question 26

Metagenome

Answer 26

all genetic information from a group of organisms. (eg gut flura, skin bacteria). Can influence obesity, disease susceptibility and is influenced by sex and body polarity

Question 27

Variant of Unknown Significance

Answer 27

Coding variant without an associated functional annotation (e.g., disrupts protein function, stabilizes mRNA)

Question 28

deep mutational scanning

Answer 28

Mutagenesis provides insight into proteins, but only recently have assays that couple genotype to phenotype been used to assess the activities of as many as 1 million mutant versions of a protein in a single experiment. This approach—’deep mutational scanning’—yields large-scale data sets that can reveal intrinsic protein properties, protein behavior within cells and the consequences of human genetic variation.

Question 29

V(D)J recombination

Answer 29

a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination takes place in the primary lymphoid tissue (the bone marrow for B cells, and Thymus for T cells). The initial steps of VDJ recombination are carried out by recombination-activating gene-1 and -2 (Rag1 and Rag2), which leads to NHEJ repair.

Question 30

Repair of DSB by non-homologous end joining (NHEJ): overview

Answer 30

1) NHEJ initiates with recognition of the DS break by Ku 2) Ku recruits DNA-PKcs 3) NHEJ variably uses combinations of: nuclease to remove the damaged DNA if there is damage
(Ku, DNA-PKcs + Artemis complex), polymerase to fill gaps, repair finishes with ligase to restore the continuous phosphodiester backbone on both strands (LIG-4)

Question 31

DNA-PKcs

Answer 31

belongs to the phosphatidylinositol 3-kinase-related kinase protein family. DNA-PKcs is the catalytic subunit of a nuclear DNA-dependent serine/threonine protein kinase called DNA-PK. The second component is the autoimmune antigen Ku. On its own, DNA-PKcs is inactive and relies on Ku to direct it to DNA ends and trigger its kinase activity DNA-PKcs is required for the non-homologous end joining (NHEJ) pathway of DNA repair, which rejoins double-strand breaks. It is also required for V(D)J recombination, a process that utilizes NHEJ to promote immune system diversity. It induces a conformational change that allows end processing enzymes to access the ends of the double-strand break.[3] DNA-PK also cooperates with ATR and ATM to phosphorylate proteins involved in the DNA damage checkpoint.

Question 32

Ku

Answer 32

a protein that binds to DNA double-strand break ends and is required for the non-homologous end joining (NHEJ) pathway of DNA repair. Ku is evolutionarily conserved from bacteria to human. Ku forms a complex with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the full DNA-dependent protein kinase, DNA-PK. Ku is thought to function as a molecular scaffold to which other proteins involved in NHEJ can bind.

Question 33

Artemis complex

Answer 33

has single-strand-specific 5’ to 3’ exonuclease activity, but it can also complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) to gain endonuclease activity on hairpins and the 5’ and 3’ overhangs; the DNA-PKcs phosphorylates Artemis to give it this new function. The four ends of DNA (two hairpinned coding ends and the two signal ends) are held together in a postcleavage complex by the RAG complex. The Artemis:DNA-PKcs complex, along with Ku and DNA ligase IV/XRCC4 dimer, can then close up the signal ends into a ‘signal joint’. It also opens the hairpins of the coding ends, and this process is thought to be mediated by the RAG complex (the RAG complex can open free hairpins by itself, but this is only observed in manganese-containing buffers, and not in magnesium-containing buffers). Nucleotides are added at the open ends by terminal deoxynucleotidyl transferase (TdT). This occurs until there are complimentary sequences at which point the opposite strands will pair up. Exonucleases then remove the unpaired nucleotides, and ligases fill in the gaps. This creates a junction between each joined segment, containing an unspecified number of nucleotide additions, flanked by a 2-residue palindromic sequence.

Question 34

resection during DSB

Answer 34

After a double-strand break occurs, sections of DNA around the 5’ ends of the break are cut away in a process

Question 35

Strand invasion

Answer 35

an overhanging 3’ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. In cells that divide through mitosis, the recipient DNA duplex is generally a sister chromatid, which is identical to the damaged DNA molecule and provides a template for repair. In meiosis, however, the recipient DNA tends to be from a similar but not necessarily identical homologous chromosome.[25] A displacement loop (D-loop) is formed during strand invasion between the invading 3’ overhang strand and the homologous chromosome. After strand invasion, a DNA polymerase extends the end of the invading 3’ strand by synthesizing new DNA. This changes the D-loop to a cross-shaped structure known as a Holliday junction. Following this, more DNA synthesis occurs on the invading strand (i.e., one of the original 3’ overhangs), effectively restoring the strand on the homologous chromosome that was displaced during strand invasion. After the stages of resection, strand invasion and DNA synthesis, the DSBR and SDSA pathways become distinct.

Question 36

synthesis-dependent strand annealing (SDSA) pathway

Answer 36

Homologous recombination via the SDSA pathway occurs in cells that divide through mitosis and meiosis and results in non-crossover products. In this model, the invading 3’ strand is extended along the recipient DNA duplex by a DNA polymerase, and is released as the Holliday junction between the donor and recipient DNA molecules slides in a process called branch migration. The newly synthesized 3’ end of the invading strand is then able to anneal to the other 3’ overhang in the damaged chromosome through complementary base pairing. After the strands anneal, a small flap of DNA can sometimes remain. Any such flaps are removed, and the SDSA pathway finishes with the resealing, also known as ligation, of any remaining single-stranded gaps. The SDSA pathway produces non-crossover recombinants. During meiosis non-crossover recombinants also occur frequently and these appear to arise mainly by the SDSA pathway as well. Non-crossover recombination events occurring during meiosis likely reflect instances of repair of DNA double-strand damages or other types of DNA damages.

Question 37

double-strand break repair (DSBR) pathway

Answer 37

The DSBR pathway is unique in that the second 3’ overhang (which was not involved in strand invasion) also forms a Holliday junction with the homologous chromosome. The double Holliday junctions are then converted into recombination products by nicking endonucleases, a type of restriction endonuclease which cuts only one DNA strand. The DSBR pathway commonly results in crossover, though it can sometimes result in non-crossover products; the ability of a broken DNA molecule to collect sequences from separated donor loci was shown in mitotic budding yeast using plasmids or endonuclease induction of chromosomal events. Because of this tendency for chromosomal crossover, the DSBR pathway is a likely model of how crossover homologous recombination occurs during meiosis.

Question 38

spo11

Answer 38

creates the DSB during meiosis, which is repaired with HR

Question 39

53bp1

Answer 39

an important regulator of the cellular response to DSBs that promotes the end-joining of distal DNA ends, which is induced during V(D)J and class switch recombination as well as during the fusion of deprotected telomeres. New insights have been gained into the mechanisms underlying the recruitment of 53BP1 to damaged chromatin and how 53BP1 promotes non-homologous end-joining-mediated DSB repair while preventing homologous recombination. From these studies, a model is emerging in which 53BP1 recruitment requires the direct recognition of a DSB-specific histone code and its influence on pathway choice is mediated by mutual antagonism with breast cancer 1 (BRCA1). This protein prevents resection during G1 (leading to NHEJ) and allows it during S/G2 (leading to HR).

Question 40

Which pathway does exgenous caused DSB take?

Answer 40

DSBs caused by exogenous can either be repaired with NHEJ or HR

Question 41

RAG

Answer 41

RAG-1 and RAG-2 are proteins at the ends of VDJ genes that separate, shuffle, and rejoin the VDJ genes during NHEJ