DNA Replication Initiation Sites in Mammalian Cells

PROJECT SUMMARY / ABSTRACT Repetitive sequences create difficult-to-replicate (DTR) regions that can stall replication, threatening genomic integrity. Ribosomal DNA (rDNA), centromeric alpha (α) satellites and telomeres are repetitive DTR regions whose sequences form structures that impede replicative DNA Polymerases (Pols). The goal of this project is to gain insights into the factors that affect the accurate replication of these distinct classes of biologically important repetitive elements. We propose to examine factors that can impact their replication and key mechanisms that allow these chromosomal elements to be stably maintained. In Aim 1 we will investigate the role of translesion synthesis (TLS) Pols in replicating rDNA, α-satellite and telomere DTR regions. Evidence implicating TLS Pols in the maintenance of repetitive DTR loci strongly suggests TLS Pols play a role in enabling normal replication of repetitive DTR sequences, likely through replicative Pol – TLS Pol exchange. We will determine the extent of replicative Pol – TLS Pol exchange during replication at the single molecule level in individual live cells using a cutting-edge novel super resolution microscopy approach we have developed. We will determine the dynamics of polymerase exchange at rDNA, α-satellite and telomere loci and establish requirements for exchange including the involvement of PCNA and its monoubiquitination. Our hypothesis that TLS Pols participate in the normal replication of rDNA, α-satellite and telomere regions predicts that these polymerases consequently maintain the stability of these loci. We will test this prediction by determining if reduced TLS Pol activity compromises the stability of these DTR regions in unstressed cells. In Aim 2, we will elucidate the replication programs of the human rDNA and α-satellite repeated sequences and determine features that impact their replication. We will employ naturally occurring repeat loci and ectopically introduced chromosomes carrying defined and uniquely distinguishable human rDNA or centromere α-satellite repeats as model loci to establish specific replication programs. Using these model loci, we will determine the contribution of rDNA sequence/repeat arrangement on their replication program and analyze repeat stability in these loci to establish whether specific sequences/arrangements are more prone to instability. We will also investigate the potential impact of the microtubule binding protein tau on rDNA replication, as its binding to rDNA has been shown to be involved in replication-mediated rDNA instability. Tau also binds to centromeric α- satellite DNA, potentially affecting its replication and stability. Thus, we will determine the effect of tau expression on rDNA and α-satellite replication programs. We expect that these proposed studies will both greatly increase our understanding of rDNA, α-satellite and telomere replication and allow us to establish new paradigms for their replication. Furthermore, with TLS Pols currently being evaluated as druggable targets, particularly in cancer treatment, our studies will provide essential insight on the unanticipated impact of TLS Pol targeting on rDNA, α-satellite and telomere replication and stability.