Near-continuously synthesized leading strands in
            <i>Escherichia coli</i>
            are broken by ribonucleotide excision

Cronan, Glen E.; Kouzminova, Elena A.; Kuzminov, Andrei

doi:10.1073/pnas.1814512116

Cited by 23 publications

(29 citation statements)

References 81 publications

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“…A recent study has revisited the questions surrounding the origin of replication intermediates in bacteria ( 81 ). Surprisingly, nearly all of the LMW leading strand products observed in earlier studies can be explained by fragmentation as part of excision-repair processes.…”

Section: Introduction: the Eukaryotic Dna Replication Machinerymentioning

confidence: 99%

Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication

Bainbridge

Teague

Doherty

2021

Nucleic Acids Research

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To bypass a diverse range of fork stalling impediments encountered during genome replication, cells possess a variety of DNA damage tolerance (DDT) mechanisms including translesion synthesis, template switching, and fork reversal. These pathways function to bypass obstacles and allow efficient DNA synthesis to be maintained. In addition, lagging strand obstacles can also be circumvented by downstream priming during Okazaki fragment generation, leaving gaps to be filled post-replication. Whether repriming occurs on the leading strand has been intensely debated over the past half-century. Early studies indicated that both DNA strands were synthesised discontinuously. Although later studies suggested that leading strand synthesis was continuous, leading to the preferred semi-discontinuous replication model. However, more recently it has been established that replicative primases can perform leading strand repriming in prokaryotes. An analogous fork restart mechanism has also been identified in most eukaryotes, which possess a specialist primase called PrimPol that conducts repriming downstream of stalling lesions and structures. PrimPol also plays a more general role in maintaining efficient fork progression. Here, we review and discuss the historical evidence and recent discoveries that substantiate repriming as an intrinsic replication restart pathway for maintaining efficient genome duplication across all domains of life.

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Section: Introduction: the Eukaryotic Dna Replication Machinerymentioning

confidence: 99%

Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication

Bainbridge

Teague

Doherty

2021

Nucleic Acids Research

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“…If RNase H2 is unable to remove these ribonucleotides (19), they can have both favorable (20,21) and lethal biological consequences (22). Recent investigations have shown that ribonucleotides are deleterious lesions that can lead to mutations, large deletions, replication stress, strand breaks (topoisomerase-1 mediated repair), chromosomal rearrangements, loss of hereditary information, and disruption of transcription processes (1,(23)(24)(25)(26).…”

Section: Introductionmentioning

confidence: 99%

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

Ghodke

Guengerich

2020

Journal of Biological Chemistry

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Incorporation of ribonucleotides into DNA can severely diminish genome integrity. However, how ribonucleotides instigate DNA damage is poorly understood. In DNA, they can promote replication stress and genomic instability and have been implicated in several diseases. We report here the impact of the ribonucleotide rATP and of its naturally occurring damaged analog 1,N6-ethenoadenosine (1,N6-ϵrA) on translesion synthesis (TLS), mediated by human DNA polymerase η (hpol η), and on RNase H2–mediated incision. Mass spectral analysis revealed that 1,N6-ϵrA in DNA generates extensive frameshifts during TLS, which can lead to genomic instability. Moreover, steady-state kinetic analysis of the TLS process indicated that deoxypurines (i.e. dATP and dGTP) are inserted predominantly opposite 1,N6-ϵrA. We also show that hpol η acts as a reverse transcriptase in the presence of damaged ribonucleotide 1,N6-ϵrA but has poor RNA primer extension activities. Steady-state kinetic analysis of reverse transcription and RNA primer extension showed that hpol η favors the addition of dATP and dGTP opposite 1,N6-ϵrA. We also found that RNase H2 recognizes 1,N6-ϵrA but has limited incision activity across from this lesion, which can lead to the persistence of this detrimental DNA adduct. We conclude that the damaged and unrepaired ribonucleotide 1,N6-ϵrA in DNA exhibits mutagenic potential and can also alter the reading frame in an mRNA transcript because 1,N6-ϵrA is incompletely incised by RNase H2.

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“…Another possibility for the retention of the backbone sugars of RNA within DNA is the incomplete removal of stretches of RNA from Okazaki fragments during genome duplication (12). In Escherichia coli, ribonucleotides can increase the leading strand fragmentation during the replication process (19). The presence of ribonucleotides in DNA has been linked to systemic autoimmunity (20).…”

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confidence: 99%

Human DNA polymerase η has reverse transcriptase activity in cellular environments

Ghodke

Egli

et al. 2019

Journal of Biological Chemistry

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Edited by Patrick SungClassical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol (hpol ) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol , could not add rNTPs to a DNA primer, but the expression of transfected hpol in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol , , or -were individually deleted. These results suggest that hpol is one of the major reverse transcriptases involved in physiological processes in human cells.

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Near-continuously synthesized leading strands in Escherichia coli are broken by ribonucleotide excision

Cited by 23 publications

References 81 publications

Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication

Repriming DNA synthesis: an intrinsic restart pathway that maintains efficient genome replication

Impact of 1,N6-ethenoadenosine, a damaged ribonucleotide in DNA, on translesion synthesis and repair

Human DNA polymerase η has reverse transcriptase activity in cellular environments

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