Double strand break repair pathways promoting genetic instability

Sreejith Ramakrishnan, Purdue University

Abstract

Double strand breaks (DSBs) occur frequently in DNA from various exogenous and endogenous sources. DSBs are a serious threat to the integrity of genome and to the survival of the cells. Therefore repairing DSBs is important. However, some DSBs repair pathways can lead to deleterious consequences, including genetic instability. One such dangerous pathway is break induced replication (BIR), which repairs broken chromosomes containing only one repairable end, similar to those generated by eroded telomeres, or during collapse of a replication fork, reviewed in (McEachern and Haber, 2006, Llorente et al., 2008). BIR is initiated by invasion of the broken chromosome into a homologous template, followed by copying of hundreds of kilo-bases of DNA from the donor. The DNA synthesis during BIR leads to various genetic instabilities including frequent mutations (Deem et al., 2011), loss of heterozygosity (Bosco and Haber, 1998, Malkova et al., 2005), telomere maintenance in the absence of telomerase (Lydeard et al., 2007), and copy number variation (Lee et al., 2007, Payen et al., 2008, Hastings et al., 2009), all hallmarks of human cancer. Although, BIR is an important DSB repair pathway, little is known about the DNA synthesis during BIR. The first goal of the research presented in this thesis was to determine the mode of DNA synthesis associated with BIR and to characterize its molecular intermediates. We discovered that the DNA synthesis during BIR is significantly different from S-phase replication. We demonstrated that BIR is carried out by a migrating bubble instead of a bona-fide replication fork, leading to conservative inheritance of newly synthesized DNA. Importantly, single strand DNA accumulates behind the BIR bubble as a result of asynchrony between leading and lagging strand synthesis. The molecular mechanism of BIR that we discovered allowed us to explain all genetic instabilities resulting from BIR, including mutagenesis and various chromosomal rearrangements. Based on the importance of BIR for cancer initiation (Mai et al., 1996, Felsher and Bishop, 1999, Nagaraju et al., 2006, Stephens et al., 2011), we expect that our results will contribute towards the understanding of tumorigenesis. The other important goal of this research was to determine how DSB repair can be channeled into BIR and other deleterious pathways. We demonstrated that inverted DNA repeats (IRs), located in the vicinity of DSB site can re-route DSB repair from conservative GC pathway, into BIR and chromosome loss. This re-routing occurs because IRs channel DSB repair into inter- molecular single strand annealing (SSA), leading to the formation of inverted dicentric (ID) chromosomes, or intra- molecular SSA, leading to formation of fold-backs (FB). Processing of these secondary structures leads to additional chromosome breakage, which leads to BIR and chromosome loss. We identified the proteins participating in the formation and processing of these secondary structures and characterized the parameters affecting their formation. Since IRs are abundant in human genome, our findings will be important for understanding genetic instabilities promoted by IRs in human diseases.

Degree

Ph.D.

Advisors

Lees, Purdue University.

Subject Area

Biology|Molecular biology|Genetics

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