Helicase Promotes Replication Re-initiation from an RNA Transcript

AbstractTo ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind the DNA, resulting in a gap in the newly replicated DNA. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances the fork, the DNAP also moves forward processively, via its association with the helicase. The presence of the DNAP, in turn, increases both helicase’s processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase’s novel role in replication re-initiation, independent of replication restart proteins or primase.

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Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

Canadian Journal of Biochemistry ◽

10.1139/o81-055 ◽

1981 ◽

Vol 59 (6) ◽

pp. 396-403 ◽

Cited By ~ 2

Author(s):

Peter R. Ganz ◽

Gyorgy B. Kiss ◽

Ronald E. Pearlman

Keyword(s):

Rna Polymerase ◽

Dna Synthesis ◽

Dna Polymerase ◽

Ribosomal Rna ◽

Replication Proteins ◽

General Applicability ◽

In Vitro Synthesis ◽

Restriction Fragments ◽

Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

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DNA polymerase Gp90 activities and regulations on strand displacement DNA synthesis revealed at single‐molecule level

The FASEB Journal ◽

10.1096/fj.202100033rr ◽

2021 ◽

Vol 35 (5) ◽

Author(s):

Shuming Zhang ◽

Xue Xiao ◽

Jingwei Kong ◽

Ke Lu ◽

Shuo‐Xing Dou ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Single Molecule ◽

Strand Displacement ◽

Single Molecule Level

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Single-molecule binding characterization of primosomal protein PriA involved in replication restart

Physical Chemistry Chemical Physics ◽

10.1039/d1cp00638j ◽

2021 ◽

Author(s):

Tzu-Yu Lee ◽

Yi-Ching Li ◽

Min-Guan Lin ◽

Chwan-Deng Hsiao ◽

Hung-Wen Li

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Single Molecule ◽

Replication Forks ◽

Bacterial Survival ◽

Dna Damages ◽

Replication Restart

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal...

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Transcriptional Modulator NusA Interacts with Translesion DNA Polymerases in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00941-08 ◽

2008 ◽

Vol 191 (2) ◽

pp. 665-672 ◽

Cited By ~ 41

Author(s):

Susan E. Cohen ◽

Veronica G. Godoy ◽

Graham C. Walker

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Dna Synthesis ◽

Dna Polymerase ◽

Temperature Sensitivity ◽

Dna Polymerases ◽

Rna Polymerases ◽

Translesion Dna Synthesis ◽

Catalytic Activities ◽

Translesion Dna

ABSTRACT NusA, a modulator of RNA polymerase, interacts with the DNA polymerase DinB. An increased level of expression of dinB or umuDC suppresses the temperature sensitivity of the nusA11 strain, requiring the catalytic activities of these proteins. We propose that NusA recruits translesion DNA synthesis (TLS) polymerases to RNA polymerases stalled at gaps, coupling TLS to transcription.

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Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms

Nucleic Acids Research ◽

10.1093/nar/gkv547 ◽

2015 ◽

Vol 43 (12) ◽

pp. 5998-6008 ◽

Cited By ~ 17

Author(s):

Geraint W. Evans ◽

Johannes Hohlbein ◽

Timothy Craggs ◽

Louise Aigrain ◽

Achillefs N. Kapanidis

Keyword(s):

Dna Synthesis ◽

Real Time ◽

Dna Polymerase ◽

Single Molecule ◽

Single Molecule Studies

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Substrate conformational dynamics facilitate structure-specific recognition of gapped DNA by DNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gkz797 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10788-10800 ◽

Cited By ~ 12

Author(s):

Timothy D Craggs ◽

Marko Sustarsic ◽

Anne Plochowietz ◽

Majid Mosayebi ◽

Hendrik Kaju ◽

...

Keyword(s):

Dna Binding ◽

Dna Polymerase ◽

Single Molecule ◽

Conformational Dynamics ◽

Coarse Grained ◽

General Mechanism ◽

Binary Complex ◽

Specific Recognition ◽

Gapped Dna ◽

Dna Substrate

Abstract DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets; some proteins recognise specific DNA sequences, while others interact with specific DNA structures. While sequence-specific DNA binding has been studied extensively, structure-specific recognition mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I Klenow Fragment (Pol) substrates both alone and in DNA–Pol complexes. Using a docking approach based on a network of 73 distances collected using single-molecule FRET, we determined a novel solution structure of the single-nucleotide-gapped DNA–Pol binary complex. The structure resembled existing crystal structures with regards to the downstream primer-template DNA substrate, and revealed a previously unobserved sharp bend (∼120°) in the DNA substrate; this pronounced bend was present in living cells. MD simulations and single-molecule assays also revealed that 4–5 nt of downstream gap-proximal DNA are unwound in the binary complex. Further, experiments and coarse-grained modelling showed the substrate alone frequently adopts bent conformations with 1–2 nt fraying around the gap, suggesting a mechanism wherein Pol recognises a pre-bent, partially-melted conformation of gapped DNA. We propose a general mechanism for substrate recognition by structure-specific enzymes driven by protein sensing of the conformational dynamics of their DNA substrates.

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A Single Molecule FRET Study of Formation of RNA Polymerase II Elongation Complex on Passivated Surface

Journal of the Chinese Chemical Society ◽

10.1002/jccs.201000074 ◽

2010 ◽

Vol 57 (3B) ◽

pp. 514-521 ◽

Cited By ~ 1

Author(s):

Yen-Chen Lin ◽

Bo-Lin Lin ◽

Tommy Setiawan ◽

Chia-Chi Chang ◽

Chi-Fu Yen ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Elongation Complex ◽

Single Molecule Fret

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Single-molecule view of coordination in a multi-functional DNA polymerase

10.1101/2020.08.12.248633 ◽

2020 ◽

Author(s):

Raymond F. Pauszek ◽

Rajan Lamichhane ◽

Arishma Rajkarnikar Singh ◽

David P. Millar

Keyword(s):

Dna Polymerase ◽

Single Molecule ◽

Active Sites ◽

Excision Repair ◽

Product Formation ◽

Pol I ◽

Key Enzyme ◽

Functional Dna ◽

Base Excision ◽

Dna Substrate

AbstractReplication and repair of genomic DNA requires the action of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from E. coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5’ flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase (pol) and 5’ nuclease (5’ nuc) domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5’ nuc domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.

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Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA

10.1101/684738 ◽

2019 ◽

Cited By ~ 1

Author(s):

Wooyoung Kang ◽

Kook Sun Ha ◽

Heesoo Uhm ◽

Kyuhyong Park ◽

Ja Yil Lee ◽

...

Keyword(s):

Rna Polymerase ◽

Single Molecule ◽

Sigma Factor ◽

Single Molecule Fluorescence ◽

Fluorescence Study ◽

Transcription Reinitiation ◽

Dna Template ◽

Rna Transcript

(Abstract)Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from DNA template in bacterial intrinsic termination of transcription much more often than concurrent dissociation. As termination is defined by release of product RNA from transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as ‘recycling.’ During the recycling stage, RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and these RNAPs can initiate transcription again at nearby promoters in case of retaining a sigma factor. The efficiency of this event, termed here as ‘reinitiation,’ increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on DNA template for reinitiation most times.

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DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

10.1101/306175 ◽

2018 ◽

Author(s):

Carel Fijen ◽

Mariam Mahmoud ◽

Rebecca Kaup ◽

Jamie Towle-Weicksel ◽

Joann Sweasy ◽

...

Keyword(s):

Dna Repair ◽

Dna Polymerase ◽

Single Molecule ◽

Conformational Changes ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dna Polymerase Β ◽

Gapped Dna ◽

Dna Substrate

The eukaryotic DNA polymerase β plays an important role in cellular DNA repair as it fills gaps in single nucleotide gapped DNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially substrate binding and a fidelity-related conformational change called fingers closing. Here, we applied single-molecule Förster resonance energy transfer to study the conformational dynamics of Pol β. Using an acceptor labelled polymerase and a donor labelled DNA substrate, we measured distance changes associated with DNA binding and fingers movement. Our findings suggest that Pol β does not bend its gapped DNA substrate to the extent related crystal structures indicate: instead, bending seems to be significantly less profound. Furthermore, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. Additionally, we provide evidence that the fingers close only partially when an incorrect nucleotide is bound. This ajar conformation found in Pol β, a polymerase of the X-family, suggests the existence of an additional fidelity checkpoint similar to what has been previously proposed for a member of the A-family, the bacterial DNA polymerase I.

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