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Fission yeast Swi1-Swi3 complex facilitates DNA binding of Mrc1.

ABSTRACT: Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.

SUBMITTER: Tanaka T

PROVIDER: S-EPMC3000942 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).

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Fission yeast Swi1-Swi3 complex facilitates DNA binding of Mrc1.

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