Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

Ozdemir, Ahmet Yunus; Rusanov, Timur; Kent, Tatiana; Siddique, Labiba A.; Pomerantz, Richard T.

doi:10.1074/jbc.ra117.000565

Cited by 40 publications

(38 citation statements)

References 46 publications

(74 reference statements)

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“…1) 1,10,11 . Polθ-hel is related to HELQ/Hel308 helicases, which are involved in replication and repair 1,12,13 . Recent studies show that Polθ-hel unwinds short DNA in an ATP-dependent manner with 3′-5′ polarity, similar to HELQ/Hel308 12 .…”

Section: Introductionmentioning

confidence: 99%

“…Polθ-hel is related to HELQ/Hel308 helicases, which are involved in replication and repair 1,12,13 . Recent studies show that Polθ-hel unwinds short DNA in an ATP-dependent manner with 3′-5′ polarity, similar to HELQ/Hel308 12 . Polθ-hel also facilitates annealing of complementary ssDNA in an ATP-independent manner, and anneals RPA coated ssDNA by utilizing the energy of ATP 14 .…”

Section: Introductionmentioning

confidence: 99%

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Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

et al. 2019

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DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

et al. 2019

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“…Pol theta also contains an ATPase-dependent SF2 helicase domain at its N terminus. This domain has been shown to unwind short pieces of duplex DNA [38] and remove replication protein A (RPA) from ssDNA [39]. The helicase domain can also anneal complementary ssDNA ends [19], consistent with its role in promoting TMEJ.…”

Section: The Mechanism Of Tmejmentioning

confidence: 81%

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution

Hanscom

McVey

2020

Cells

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Double-strand breaks are one of the most deleterious DNA lesions. Their repair via error-prone mechanisms can promote mutagenesis, loss of genetic information, and deregulation of the genome. These detrimental outcomes are significant drivers of human diseases, including many cancers. Mutagenic double-strand break repair also facilitates heritable genetic changes that drive organismal adaptation and evolution. In this review, we discuss the mechanisms of various error-prone DNA double-strand break repair processes and the cellular conditions that regulate them, with a focus on alternative end joining. We provide examples that illustrate how mutagenic double-strand break repair drives genome diversity and evolution. Finally, we discuss how error-prone break repair can be crucial to the induction and progression of diseases such as cancer.

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“…More surprisingly, we demonstrated that nsP13 is more favorable on DNA in the early stages of reaction initiation and binds DNA substrates faster than RNA substrates of the same sequence. HCV NS3 RNA helicase also showed poor processivity on RNA than DNA 31 , and some observations revealed that the loading strand of the hybrid substrate was chosen for the unwinding efficiency of the helicase in the single-turnover condition 47,49 . For these reasons, although RNA helicase nsP13 can unwind both duplex RNA and DNA, their different affinities for RNA and DNA may also be a key factor to determine nucleic acid unwinding and translocation by nsP13 in unwinding of the duplex.…”

Section: Discussionmentioning

confidence: 99%

A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA

Jang

Jeong

Kang

et al. 2020

Sci Rep

102

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Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5′ single-stranded tail in a 5′ to 3′ direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5′-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased Atps to processively unwind duplex RnA. our results suggest that duplex RnA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease caused by a novel coronavirus (SARS-CoV or SCV) that has claimed almost 800 deaths in early 2003 1 . SCV is an enveloped, positive single-stranded RNA virus (or (+) ssRNA virus) with a genome of ~30 kb in length 2,3 . Two-thirds of the SCV genome at the 5′-end comprise replicase genes (orf1ab) encoding 16 nonstructural proteins (nsPs). The replicase genes comprising open reading frames (OFR1a and 1b) are translated into two large replicative polyproteins, pp1ab (~790 kDa) and pp1a (~490 kDa), which are involved with and without ribosomal frameshifting into the −1 frame 4,5 . These two translational polyproteins are processed autoproteolytically by the major viral cysteine proteases M PRO or 3CL PRO to produce 16 non-structural proteins (nsPs), including RNA-dependent RNA polymerases (RdRp, nsP12) and NTPase/helicase (nsP13) 6-8 . These viral replicases are the core of membrane-bound replication-transcription complexes that synthesize the entire viral genome and eight subgenomic mRNAs 9,10 .Because viral helicase is considered to be essential for subsequent viral replication and proliferation, it is an important potential target for antiviral therapy [11][12][13] . In addition, the inhibition of these targets may interfere with the metabolism of the infecting virus without strong side effects in patients. Several viral helicases have been used as proven drug targets due to the inhibition of helicase activity in animal models of herpes simplex virus (HSV) and in the treatment of hepatitis C 14,15 . Therefore, much effort has been spent on the development of small-molecule inhibitors and chemicals as drug candidates to inhibit the function of SARS coronavirus helicase nsP13 (SCV ns...

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Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids

Cited by 40 publications

References 46 publications

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

Molecular basis of microhomology-mediated end-joining by purified full-length Polθ

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution

A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA

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