Publication Date:
2013-03-29
Description:
Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands. However, coupled replication presents a largely unrecognized topological problem. Because DNA polymerase must travel a helical path during synthesis, the physical connection between leading- and lagging-strand polymerases causes the daughter strands to entwine, or produces extensive build-up of negative supercoils in the newly synthesized DNA. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is unknown. Here we examine the dynamics of the Escherichia coli replisome, using ensemble and single-molecule methods, and show that the replisome may solve the topological problem independent of topoisomerases. We find that the lagging-strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome because of its contact with the leading-strand polymerase. This behaviour, referred to as 'signal release', had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments. However, we observe that signal release is independent of primase and does not seem to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618558/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618558/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kurth, Isabel -- Georgescu, Roxana E -- O'Donnell, Mike E -- GM38839/GM/NIGMS NIH HHS/ -- R01 GM038839/GM/NIGMS NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2013 Apr 4;496(7443):119-22. doi: 10.1038/nature11988. Epub 2013 Mar 27.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23535600" target="_blank"〉PubMed〈/a〉
Keywords:
DNA/chemistry/genetics/metabolism
;
DNA Primase/metabolism
;
*DNA Replication
;
DNA, Bacterial/biosynthesis/chemistry/genetics/*metabolism
;
DNA, Superhelical/biosynthesis/chemistry/genetics/metabolism
;
DNA-Binding Proteins/chemistry/metabolism
;
DNA-Directed DNA Polymerase/chemistry/*metabolism
;
Escherichia coli/*enzymology/*genetics
;
Microscopy, Fluorescence
;
Multienzyme Complexes/chemistry/*metabolism
;
*Nucleic Acid Conformation
;
Protein Binding
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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