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Completion of DNA Replication in Escherichia Coli

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Completion of DNA Replication in Escherichia Coli Completion of DNA replication in Escherichia coli Brian M. Wendel1, Charmain T. Courcelle, and Justin Courcelle Department of Biology, Portland State University, Portland, OR 97201 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved September 29, 2014 (received for review August 5, 2014) The mechanism by which cells recognize and complete replicated would generate a third copy of the chromosomal region where the regions at their precise doubling point must be remarkably efficient, event occurs. Thus, over-replication may be inherent and pro- occurring thousands of times per cell division along the chromo- miscuous during the duplication of genomes. If true, then to somes of humans. However, this process remains poorly understood. ensure that each sequence of the genome replicates once, and Here we show that, in Escherichia coli, the completion of replication only once, per generation, cells must encode an enzymatic system involves an enzymatic system that effectively counts pairs and limits that is essentially able to count in pairs and efficiently degrade cellular replication to its doubling point by allowing converging rep- odd or over-replicated regions until the two nascent end pairs of lication forks to transiently continue through the doubling point replication events can be joined. before the excess, over-replicated regions are incised, resected, and The model organism E. coli is particularly well-suited to dis- joined. Completion requires RecBCD and involves several proteins sect how this fundamental process occurs. In E. coli, the com- associated with repairing double-strand breaks including, ExoI, pletion of replication occurs at a defined region on the genome, SbcDC, and RecG. However, unlike double-strand break repair, com- opposite to the bidirectional origin of replication (15). Most completion events can be further localized to one of six termi- pletion occurs independently of homologous recombination and nation (ter) sequences within the 400-kb terminus region due to RecA. In some bacterial viruses, the completion mechanism is specif- the action of Tus, which binds to ter and inhibits replication fork ically targeted for inactivation to allow over-replication to occur dur- progression in an orientation-dependent manner, in effect stalling ing lytic replication. The results suggest that a primary cause of the replication fork at this site until the second arrives (16, 17). genomic instabilities in many double-strand-break-repair mutants Although Tus confines converging replication forks to a specific arises from an impaired ability to complete replication, independent region, it does not appear to be directly involved in the completion from DNA damage. reaction because tus mutants have no phenotype and complete replication normally (18). Furthermore, plasmids and bacterio- replication completion | double-strand break repair | RecBCD | phage lacking ter sequences are maintained stably (19). homologous recombination | SbcDC Many mutants impaired for either replication initiation or elongation were initially isolated based on their growth defects or uring chromosomal replication, cells tightly regulate the an impaired ability to maintain plasmids (20–22). We reasoned Dprocesses of initiation, elongation, and completion to ensure that mutants impaired for the ability to complete replication might that each daughter cell inherits an identical copy of the genetic be expected to exhibit similar phenotypes and initially focused our information. Although the mechanisms regulating initiation and attention on the properties of recBC and recD mutants. RecB-C-D – elongation have been well characterized (reviewed in refs. 1, 2), forms a helicase nuclease complex that is required for homolo- gous repair of double-strand breaks in E. coli (23, 24). The enzyme the process of how cells recognize replicated regions and complete “ ” replication at the precise doubling point remains a fundamental uses specific DNA sequences, termed Chi sites, to initiate re- combination between pairs of molecules. Loss of RecB or C question yet to be addressed. Whether this event occurs once per inactivates the enzyme complex, whereas loss of RecD inactivates generation as in Escherichia coli or thousands of times per gen- the nuclease and Chi recognition, but retains helicase activity (23, eration as in human cells, the failure to efficiently carry out this 24). Here, we show that inactivation of RecBCD leads to a failure function would be expected to result in a loss of genomic stability. Considering the large number of proteins that cells devote to Significance ensuring the fidelity of replication initiation and elongation, it seems highly probable that the final critical step in this process will be also be tightly regulated and controlled enzymatically. All phases of DNA replication are tightly regulated to ensure that In some aspects, one could argue that the efficiency of com- daughter cells inherit a precise copy of the genomic DNA. Al- pletion is likely to be more critical to the faithful duplication of though the mechanisms regulating initiation and elongation have the genome than that of initiation. When replication origins fail been well characterized, the process of how cells recognize rep- to initiate efficiently, elongation of replication forks from licated regions and complete replication at the precise doubling neighboring origins is often able to compensate (3, 4), and both point remains a fundamental question yet to be addressed. Here prokaryotic and eukaryotic cells are able to tolerate variations in we show that the completion of replication involves a transient their origin number without severe phenotypic consequences (5– over-replication of the region where forks converge before the 7). However, a failure to accurately limit or join any event where excess regions are incised, resected, and joined. Completion forks converge would be expected to result in duplications, requires several proteins associated with repairing double-strand deletions, rearrangements, or a loss of viability depending upon breaks, but unlike break repair, it occurs independently of ho- how the DNA ends are resolved at segregation. mologous recombination and is targeted for inactivation by some A number of studies suggest that an ability to sense when all bacterial viruses during the transition to lytic replication. sequences in the genome have doubled is critical to genomic replication. In vitro, converging replisomes continue through Author contributions: B.M.W., C.T.C., and J.C. designed research; B.M.W., C.T.C., and J.C. their meeting point as one replisome displaces the other, resulting performed research; B.M.W., C.T.C., and J.C. analyzed data; and B.M.W. wrote the paper. in over-replication, or a third copy, of the region where the forks The authors declare no conflict of interest. meet (8). Complicating the process of genomic doubling even This article is a PNAS Direct Submission. further, several studies have suggested that illegitimate initiations Data deposition: The data reported in this paper have been deposited in the Sequence of replication frequently occur at single-strand nicks, gaps, D- Read Archive (SRA), www.ncbi.nlm.nih.gov/Traces/sra (accession no. SRP047195). loops, and R-loops throughout the genomes of both prokaryotes 1To whom correspondence should be addressed. Email: [email protected]. – and eukaryotes (9 14). Similar to when replication forks continue This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. through a previously replicated template, each of these events 1073/pnas.1415025111/-/DCSupplemental. 16454–16459 | PNAS | November 18, 2014 | vol. 111 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.1415025111 Downloaded by guest on September 26, 2021 to recognize and join replicating molecules at their doubling point. Although the completion process requires RecBCD, it is distinct recD AE0.8 from double-strand break repair and does not involve a double- sbcDC recF recG strand break intermediate, homologous recombination, or RecA. 0.6 wild type wild type recA xonA Results sbcDCxonA 0.4 Similar to other mutants that are involved in replication initiation recBC 0.2 or elongation, recBC and recD mutants each exhibit growth abnormalities and plasmid instabilities. These phenotypes are Absorbance (630nm) 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 unique compared to those of other recombination mutants, and Hours Hours suggest that these mutants have a broader, more fundamental BFwild type recA recBC recD recF recG sbcDCxonA function in replicating cells. Relative to wild-type cultures, recBC - + - + - + - + - + - + - + Amp cultures grow poorly and produce large numbers of small, non- viable cells, whereas recD cultures grow for a longer time period and reach a higher cell density (Fig. 1A)(25–28). By comparison, cultures lacking either RecF or RecA, which is essential for all homologous recombination and RecBCD-mediated double-strand break repair, grow comparatively well, arguing that some function of RecBCD is unique from homologous repair and DNA damage. CGMutations inactivating RecBC or RecD also affect the stability F of plasmid minichromosomes, a feature that is again distinct wild type recA recBC recD recG xonA sbcDC xonA sbcDC rec from other recombination mutants (Fig. 1B) (28, 29). Plasmids linear and circular size (kb) multimers A No UV 25 J/m2 UV post- pre- 14C DNA (CPM) trimers 36000 post- pre- 9000 23 treatment treatment treatment treatment dimers synthesis DNA synthesis DNA 9.4 nicked/gapped 6.5 monomers linear 3H DNA (CPM) 4.3 monomers Wild type 16000 800014C DNA (CPM) supercoiled monomers D recA 2.0 F 3H DNA (CPM) recA recBC recD rec wild type 1500 4000 14C DNA (CPM) linear multimers marker 8-mer 23kb 7-mer 6-mer 5-mer 4-mer recBC 9.4 3H DNA (CPM) 6.5 3-mer B Increasing density Increasing density 4.3 2-mer 6 wild recA recBC nicked and linear monomers type 5 2.3 1-mer 2.0 4 -UV Fig. 1. recBC and recD mutants exhibit growth abnormalities and an im- 3 +UV paired ability to maintain monomeric plasmids.
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