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High-Accuracy Lagging-Strand DNA Replication Mediated by DNA Polymerase Dissociation

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High-Accuracy Lagging-Strand DNA Replication Mediated by DNA Polymerase Dissociation High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation Katarzyna H. Maslowskaa,b, Karolina Makiela-Dzbenskab, Jin-Yao Moa, Iwona J. Fijalkowskab,1, and Roel M. Schaapera,1 aGenome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709; and bInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved March 2, 2018 (received for review November 27, 2017) The fidelity of DNA replication is a critical factor in the rate at Detailed mutational studies in E. coli have suggested that the − − which cells incur mutations. Due to the antiparallel orientation of in vivo polymerase misinsertion rate is approximately 10 4–10 5 the two chromosomal DNA strands, one strand (leading strand) is per base copied, and that proofreading by the e subunit reduces the replicated in a mostly processive manner, while the other (lagging error rate by approximately 102-fold, yielding an overall polymerase − − strand) is synthesized in short sections called Okazaki fragments. A error rate of 10 6–10 7 per replicated base (1). DNA mismatch fundamental question that remains to be answered is whether the repair (via the mutHLS system), which follows the replication fork, two strands are copied with the same intrinsic fidelity. In most then reduces the observed error rate by another 102-to103-fold, − experimental systems, this question is difficult to answer, as the accounting for the overall mutation rate of approximately 10 10 per replication complex contains a different DNA polymerase for each replicated base (1, 3). However, many aspects of the replisome and δ e strand, such as, for example, DNA polymerases and in eukary- its fidelity remain incompletely understood, including its precise otes. Here we have investigated this question in the bacterium composition (4, 5), the fidelity role of other HE subunits (6), the Escherichia coli , in which the replicase (DNA polymerase III holo- interference of the accessory DNA polymerases (7), and the effi- enzyme) contains two copies of the same polymerase (Pol III, the – dnaE ciency of exonucleolytic proofreading (8 10). gene product), and hence the two strands are copied by In the present work, we have addressed another fundamental the same polymerase. Our in vivo mutagenesis data indicate that question: whether the two DNA strands—leading and lagging— the two DNA strands are not copied with the same accuracy, and GENETICS are subject to the same fidelity rules. Insight into this matter may that, remarkably, the lagging strand has the highest fidelity. We also help understand the origin of DNA strand biases observed postulate that this effect results from the greater dissociative char- in studies of mutagenesis, evolution, and cancer (11–13). acter of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by Results and Discussion results with dnaE antimutator polymerases characterized by in- A System to Investigate in Vivo Chromosomal DNA Replication Fidelity. creased dissociation rates. To study any differential effects of leading and lagging-strand DNA replication fidelity | leading and lagging strands | DNA polymerase III replication on chromosomal replication fidelity, we developed holoenzyme | dnaE antimutators | DNA polymerase dissociation the system shown schematically in Fig. 1A. Mutations are scored in the 1,100-bp lacI gene, encoding the repressor of the lacZYA operon. Forward mutations occurring throughout the lacI gene, he accuracy by which organisms are able to duplicate their inactivating the repressor function, lead to constitutive expression Tchromosomal DNA is generally high, producing on average only one error for every 109–1011 copied bases. This high fidelity is not achieved in a single step, but instead is produced via the op- Significance eration of several sequential error-avoidance and editing steps. These steps include selection of the correct DNA base by the The accuracy (fidelity) by which cells are able to duplicate their DNA polymerase (i.e., nucleotide insertion step), the editing (i.e., chromosomal DNA before cell division is an important factor in removal) of polymerase misinsertion errors by exonucleolytic the frequency at which they accumulate mutations. Because proofreading, and finally postreplicative DNA mismatch repair, mutations are generally harmful, organisms have developed which detects and corrects DNA mismatches in newly repli- various mechanisms to minimize the frequency of errors during cated DNA (1). DNA replication. Replication is generally performed by large Chromosomal DNA replication is generally performed by multiple subunit complexes (replicases), which simultaneously multisubunit DNA polymerase complexes (replicases) that con- and in a coordinated fashion copy the two DNA strands. Due to duct the simultaneous, coordinated synthesis of the two new the antiparallel nature of the two DNA strands, the replication DNA strands at the replication fork. The replication speed can enzymology of the two individual strands is slightly different, be very high, up to 500–1,000 nucleotides per second for the and our study demonstrates that the two strands are copied bacterium Escherichia coli (2). In this organism, replication is with different accuracies. Specifically, the discontinuous lag- performed by the multisubunit DNA polymerase III holoenzyme ging strand is significantly more accurate than the more proc- essive leading strand. (HE) complex (2), which contains two copies of a DNA poly- merase core, one for each strand, with composition αeθ, in which α e Author contributions: I.J.F. and R.M.S. designed research; K.H.M., K.M.-D., and J.-Y.M. the subunit is the DNA polymerase and is the associated performed research; K.H.M., K.M.-D., I.J.F., and R.M.S. analyzed data; and R.M.S. wrote exonucleolytic proofreader. The two core polymerases are joined the paper. together through interactions with the τ subunit of the DnaX5 The authors declare no conflict of interest. complex. The latter also serves as a loader/unloader complex for This article is a PNAS Direct Submission. β two donut-shaped ( 2) processivity clamps that tether the two Published under the PNAS license. polymerases to the DNA. The speed of the fork is controlled 1To whom correspondence may be addressed. Email: [email protected] or schaaper@ through interactions with the DNA helicase (DnaB gene prod- niehs.nih.gov. uct) that unzips the DNA ahead of the moving fork (2). How This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. such high-speed replication machines can copy DNA with im- 1073/pnas.1720353115/-/DCSupplemental. pressive fidelity is an important question. www.pnas.org/cgi/doi/10.1073/pnas.1720353115 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 A B the opposite direction in the form of short Okazaki fragments. 5' Fig. 1B illustrates the consequences of the inversion in the way that the operon is replicated. For the R orientation, the lacI 3' 5' coding sequence (indicated in blue) is copied by the lagging- Leading L-orientaon strand mechanism, while for the L orientation, it is copied by the 3' lacI leading strand replication. Our experimental system to determine possible differences in lacI replication fidelity between the leading and lagging strands is 5' R-orientaon based on a detailed analysis of the frequencies and types of lacI Lagging mutations occurring for the two gene orientations. For example, 3' 5' the most prominent replication error made by DNA polymerases is the misincorporation of dGTP opposite a template T, denoted · 3' here as T G (template strand underlined). This event will ulti- mately be observed as an A·T → G·C transition mutation. Using the lacI coding sequence as a reference, these mutations can be C A·T G·C scored at both T sites (read as T → C) and A sites (read as A → T A 5' T A R-orientaon G G G) within this sequence, but their genesis at the two sites is Lagging different. For example, in the L orientation (Fig. 1C), the mu- 3' 5' tations scored at T sites (T → C) result from T·G errors in the Leading → G G leading strand, while those scored at A sites (A G) result from 3' A T L-orientaon A T T·G errors in the lagging strand, and the reverse is true for the R orientation (Fig. 1C). Thus, insight into the relative frequency of T·G errors during leading strand and lagging-strand replication D G·C A·T can be obtained by simply comparing the ratio of these transi- G C 5' G C R-orientaon tions at coding strand T and A sites. T T · → · Lagging Likewise, for the case of the reciprocal G C A T transitions, 3' analysis in terms of the preferred G·T mispairings can be per- 5' formed simply by comparing events at reference strand G and Leading T T strand C. Referring to Fig. 1D, for the L orientation, G·C → A·T 3' C G L-orientaon C G observed at coding strand G sites (G → A) correspond to G·T errors in the leading strand, while those observed at C sites (C → Fig. 1. A system for assaying differential replication fidelity in leading and T) correspond to G·T in the lagging strand, and vice versa for the lagging strands. (A) E. coli chromosome with the lac operon inserted at the R orientation. A caveat for this overall approach is that A·T → attL locus in either the R or the L orientation. Also shown are the OriC origin, G·C mutations can alternatively result from A·C mispairings, while where bidirectional replication begins (the two arrowheads), and the TerC · → · · terminator region, where the two forks eventually meet.
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