GENOME-DESTABILIZING AND MUTAGENIC EFFECTS OF BREAK-INDUCED REPLICATION IN SACCHAROMYCES CEREVISIAE

[...]...ix PIKK phosphoinositol-3-kinase-related kinase RDR Recombination-dependent replication RNR Ribonucleotide reductase SDSA Synthesis-dependent strand annealing SSA Single-strand annealing ssDNA Single-stranded DNA x ABSTRACT Deem, Angela Kay Ph.D., Purdue University, May 2011 Genome-destabilizing and Mutagenic Effects of Break-induced Replication in Saccharomyces cerevisiae Major Professor: Cynthia... cutting of the flap, especially when the direct repeats being used for annealing are short (Sugawara et al., 1997) DNA synthesis during BIR differs from that during SSA and GC in that it is believed to involve formation of a replication fork with both leading- and lagging- 18 strand synthesis Support for formation of a processive replication fork during BIR is three-pronged First, initiation of BIR replication. .. Replication In contrast to GC, break-induced replication (BIR) repairs DSBs in which only one side of the DSB is able to participate in HR (Figure 2.1) This phenomenon was first proposed to explain the mechanism of host infection by a linear chromosome employed by bacteriophage T4 (Luder and Mosig, 1982) Subsequent studies confirmed that the ends of the infecting chromosome indeed initiated replication in an... 12 2.2.3 Single-strand Annealing Single-strand annealing (SSA) is an efficient repair pathway that repairs a DSB by annealing of complementary sequences on either side of the lesion (Figure 2.1; (Fishman-Lobell and Haber, 1992); reviewed in (Aguilera A., 2007; Paques and Haber, 1999)) Annealing of the repeated sequences requires that they first become single-stranded and, thus, the kinetics of this process... described in Chapter 3 Data obtained during this research are presented in two chapters, each of which contains an independent introduction and discussion Chapter 4 summarizes findings relevant to the genome-destabilizing effects of BIR (objectives 1 through 4), and Chapter 5 summarizes findings relevant to BIR mutagenesis (objectives 5 and 6) 3 CHAPTER 2 LITERATURE REVIEW 2.1 Types of DNA Damage DNA is subjected... delay exists between the initial invasion and processive replication, though some recent findings shed light on this issue First, the one-ended invasion that occurs during BIR initiation is unstable, and multiple rounds of invasion and short DNA synthesis can occur in different templates before processive replication of a donor molecule begins (Smith et al., 2007) Also, Jain et al (2009) described... of a DSB proceeds through: 1) 5’-to-3’ resection of both sides of the break, 2) invasion by at least one of the ssDNA ends into the donor sequence, 3) new DNA synthesis using the donor molecule template, 4) dissociation of the heteroduplex molecule, 5) re-annealing of the broken 8 molecule via newly synthesized sequences, and 6) sealing of single-strand gaps (Figures 2.1, 2.2) 2.2.2 Break-induced Replication. .. damage, leading to a variety of lesions that require repair One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome Necessarily, cells have evolved a number of DSB repair mechanisms One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous... after invasion of one side of the DSB and new DNA synthesis, the duplex molecule dissociates and re-anneals to the other side of the lesion, making crossover outcomes rare (GC by SDSA) 10 genetic investigations confirmed an analogous mechanism of a single 3’-ended ssDNA invasion into a homologous sequence and extensive synthesis to the end of the donor chromosome (Bosco and Haber, 1998; Davis and Symington,... for genetic screens Because repair proteins and pathways are widely conserved among eukaryotes, investigations into DSB repair in yeast are highly relevant to the human condition, including the phenomena of aging and disease genesis 2.2 DSB Repair Pathways in S cerevisiae It is estimated that approximately 10% of S cerevisiae cells undergoing replication will incur one or more DSBs (Aguilera A., 2007) . mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase  (Pol ), as well as. Non-reciprocal translocation PCR Polymerase chain reaction PFGE Pulsed-field gel electrophoresis Pol Polymerase ix PIKK phosphoinositol-3-kinase-related kinase RDR Recombination-dependent. characterized by decreased BIR repair and increased xi loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed