Deconstructing lagging‐strand synthesis in vivo

Abstract

Half of all nuclear DNA in eukaryotic organisms must be replicated discontinuously on the lagging strand. Because eukaryotic Okazaki fragments are short – on average ~300 nt – complete replication of the human genome must require the synthesis, processing and ligation of tens of millions of Okazaki fragments.Each Okazaki Fragment is initiated by the error‐prone DNA polymerase alpha and subsequently extended by DNA polymerase delta. Upon encountering a downstream fragment, Pol delta carries out strand‐displacement synthesis coupled to nucleolytic cleavage of the 5′ end of the displaced fragment to generate a ligation‐competent nicked DNA structure. Several partially redundant nucleases have been proposed to participate in Okazaki fragment processing: the roles of these nucleases have been extensively studied in vitro, but in vivo studies have largely relied on genetic interactions without directly examining nuclease cleavage during OF processing. Here, we describe a systematic, in vivo approach to study Okazaki fragment flap processing in Saccharomyces cerevisiae, using the direct sequencing and analysis of lagging‐strand intermediates to examine both lagging‐strand biogenesis and replication‐fork movement. Additional results detailing the cell‐cycle dependence of lagging‐strand processing will also be discussed.Support or Funding InformationSupported by NIGMS R01 GM114340 and the Searle Scholars ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.