Global landscape of replicative DNA polymerase usage in the human genome

The division of labour between DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combing the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality, zones of replication initiation and termination. We confirm that transcriptional activity shapes the patterns of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co-directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

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Transcription-mediated organization of the replication initiation program across large genes sets up common fragile sites genome-wide

10.1101/714717 ◽

2019 ◽

Cited By ~ 1

Author(s):

Olivier Brison ◽

Sami EL-Hilali ◽

Dana Azar ◽

Stéphane Koundrioukoff ◽

Mélanie Schmidt ◽

...

Keyword(s):

Replication Timing ◽

S Phase ◽

Fragile Sites ◽

Replication Initiation ◽

Common Fragile Sites ◽

Large Gene ◽

Genome Wide ◽

Underlying Mechanisms ◽

Large Genes ◽

Nascent Transcripts

ABSTRACTCommon Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

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Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide

Nature Communications ◽

10.1038/s41467-019-13674-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Olivier Brison ◽

Sami El-Hilali ◽

Dana Azar ◽

Stéphane Koundrioukoff ◽

Mélanie Schmidt ◽

...

Keyword(s):

Replication Timing ◽

S Phase ◽

Fragile Sites ◽

Replication Initiation ◽

Common Fragile Sites ◽

Large Gene ◽

Genome Wide ◽

Underlying Mechanisms ◽

Large Genes ◽

Nascent Transcripts

AbstractCommon fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription–replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription–replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

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Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

10.1101/352567 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sarina Y. Porcella ◽

Natasha C. Koussa ◽

Colin P. Tang ◽

Daphne N. Kramer ◽

Priyanka Srivastava ◽

...

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Replication Fork ◽

Replication Initiation ◽

Replication Origins ◽

Origin Firing ◽

Leading Strand ◽

Strand Initiation ◽

Lagging Strand

AbstractDuring eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

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Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604894113 ◽

2016 ◽

Vol 113 (21) ◽

pp. 5916-5921 ◽

Cited By ~ 8

Author(s):

Alfredo J. Hernandez ◽

Seung-Joo Lee ◽

Charles C. Richardson

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Fragment Length ◽

Dna Binding Protein ◽

Okazaki Fragment ◽

Dna Primase ◽

Dna Strands ◽

Leading Strand ◽

Rate Limiting ◽

Lagging Strand

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

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Ribonucleotide incorporation characteristics around yeast autonomously replicating sequences reveal the labor division of replicative DNA polymerases

10.1101/2020.08.27.270728 ◽

2020 ◽

Author(s):

Penghao Xu ◽

Francesca Storici

Keyword(s):

Dna Polymerase ◽

Genome Instability ◽

Dna Polymerases ◽

Yeast Cells ◽

Yeast Genome ◽

Wild Type ◽

Specific Sequence ◽

Leading Strand ◽

Mapping Techniques ◽

Lagging Strand

ABSTRACTRibonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.

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Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase—Pol ε Interaction

International Journal of Molecular Sciences ◽

10.3390/ijms21249484 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9484

Author(s):

Milena Denkiewicz-Kruk ◽

Malgorzata Jedrychowska ◽

Shizuko Endo ◽

Hiroyuki Araki ◽

Piotr Jonczyk ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Synthetic Lethality ◽

Replication Initiation ◽

Recombination Rates ◽

Dna Polymerase Epsilon ◽

Dna Replication Initiation ◽

Leading Strand

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

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Alterations of DNA and Chromatin Structures at Telomeres and Genetic Instability in Mouse Cells Defective in DNA Polymerase α

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.11073-11088.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 11073-11088 ◽

Cited By ~ 32

Author(s):

Mirai Nakamura ◽

Akira Nabetani ◽

Takeshi Mizuno ◽

Fumio Hanaoka ◽

Fuyuki Ishikawa

Keyword(s):

Telomere Length ◽

Dna Polymerase ◽

Temperature Sensitive ◽

Telomeric Dna ◽

Dna Polymerase Α ◽

Genome Wide ◽

Associated Proteins ◽

Mouse Cells ◽

The Impact ◽

Lagging Strand

ABSTRACT Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase α, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase α activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase α mutant allele. When polymerase α was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase α inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase α is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.

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Transcription-dependent regulation of replication dynamics modulates genome stability

10.1101/286807 ◽

2018 ◽

Author(s):

Marion Blin ◽

Benoît Le Tallec ◽

Viola Naehse ◽

Mélanie Schmidt ◽

Gael A. Millot ◽

...

Keyword(s):

Genome Stability ◽

Chromosomal Rearrangements ◽

Replication Timing ◽

Replication Stress ◽

S Phase ◽

Fragile Sites ◽

Replication Initiation ◽

Replication Gene ◽

Large Genes ◽

Dynamics Analyses

Replication stress is a primary threat to genome stability and has been implicated in tumorigenesis1, 2. Common fragile sites (CFSs) are loci hypersensitive to replication stress3 and are hotspots for chromosomal rearrangements in cancers4. CFSs replicate late in S-phase3, are cell-type dependent4–6 and nest within very large genes4, 7–9. The mechanisms responsible for CFS instability are still discussed, notably the relative impact of transcription-replication conflicts7, 8, 10versus their low density in replication initiation events5, 6. Here we address the relationships between transcription, replication, gene size and instability by manipulating the transcription of three endogenous large genes, two in chicken and one in human cells. Remarkably, moderate transcription destabilises large genes whereas high transcription levels alleviate their instability. Replication dynamics analyses showed that transcription quantitatively shapes the replication program of large genes, setting both their initiation profile and their replication timing as well as regulating internal fork velocity. Noticeably, high transcription levels advance the replication time of large genes from late to mid S-phase, which most likely gives cells more time to complete replication before mitotic entry. Transcription can therefore contribute to maintaining the integrity of some difficult-to-replicate loci, challenging the dominant view that it is exclusively a threat to genome stability.

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DNA polymerase η contributes to genome-wide lagging strand synthesis

Nucleic Acids Research ◽

10.1093/nar/gky1291 ◽

2018 ◽

Vol 47 (5) ◽

pp. 2425-2435 ◽

Cited By ~ 7

Author(s):

Katrin Kreisel ◽

Martin K M Engqvist ◽

Josephine Kalm ◽

Liam J Thompson ◽

Martin Boström ◽

...

Keyword(s):

Dna Polymerase ◽

Genome Wide ◽

Lagging Strand

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Inversion of asymmetric histone deposition upon replication stress

10.1101/2021.04.20.440573 ◽

2021 ◽

Author(s):

Frank J Dekker ◽

Martijn Zwinderman ◽

Thamar Jessurun Lobo ◽

Petra Van der Wouden ◽

Diana C.J. Spierings ◽

...

Keyword(s):

Molecular Mechanisms ◽

Replication Stress ◽

Wide Distribution ◽

Replication Initiation ◽

Sister Chromatids ◽

Genome Wide ◽

Histone Deposition ◽

Dna Template ◽

P53 Inactivation ◽

Lagging Strand

Following DNA replication, equal amounts of histones are distributed over sister chromatids by re-deposition of parental histones and deposition of newly synthesized histones. Molecular mechanisms balancing the allocation of new and old histones remain largely unknown. Here, we studied the genome-wide distribution of new histones relative to parental DNA template strands and replication initiation zones using double-click-seq. In control conditions, new histones were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment, and inhibition of ATR or p53 inactivation, inverted the observed histone deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on RPA occupancy. We propose that asymmetric deposition of newly synthesized histones onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

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