Abstract
SummaryThe sliding clamp PCNA is a crucial component of the DNA replication machinery. Timely PCNA loading and unloading are central for genome integrity and must be strictly coordinated with other DNA processing steps during replication. Here, we show that the S. cerevisiae Elg1 replication factor C-like complex (Elg1-RLC) unloads PCNA genome-wide following Okazaki fragment ligation. In the absence of Elg1, PCNA is retained on chromosomes in the wake of replication forks, rather than at specific sites. Degradation of the Okazaki fragment ligase Cdc9 leads to PCNA accumulation on chromatin, similar to the accumulation caused by lack of Elg1. We demonstrate that Okazaki fragment ligation is the critical prerequisite for PCNA unloading, since Chlorella virus DNA ligase can substitute for Cdc9 in yeast and simultaneously promotes PCNA unloading. Our results suggest that Elg1-RLC acts as a general PCNA unloader and is dependent upon DNA ligation during chromosome replication.
Highlights
Integrity of the DNA replication machinery is crucial to ensure accurate duplication of the genetic information and subsequent transfer to daughter cells
By dissecting DNA ligase functions and examining effects of substituting yeast DNA ligase with an exogenous viral ligase, we find that unloading of PCNA by Elg1-RLC depends on completion of Okazaki fragment ligation
To investigate where on chromosomes PCNA accumulates in S. cerevisiae lacking Elg1, we carried out chromatin immunoprecipitation sequencing (ChIPseq) analysis
Summary
Integrity of the DNA replication machinery is crucial to ensure accurate duplication of the genetic information and subsequent transfer to daughter cells. PCNA encircles DNA to act as a sliding clamp, ensuring processivity of DNA polymerases, and a platform for recruitment of numerous other replication proteins (Moldovan et al, 2007). Two important components whose recruitment is assisted by direct interaction with PCNA are the flap endonuclease FEN-1 and DNA ligase I (Beattie and Bell, 2011). Both these proteins are involved in the processing of Okazaki fragments, the series of short fragment precursors first synthesized and ligated to assemble the nascent lagging strand. PCNA must be loaded on the DNA repeatedly, at the initiation of each Okazaki fragment
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