DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo

DNA Repair ◽  
2018 ◽  
Vol 64 ◽  
pp. 59-67 ◽  
Author(s):  
Kang-Yi Su ◽  
Liang-In Lin ◽  
Steven D. Goodman ◽  
Rong-Syuan Yen ◽  
Cho-Yuan Wu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Exonuclease Activity ◽  
Repair Pathway ◽  
Dna Polymerase I ◽  
Endonuclease V ◽  
Polymerase I

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

Viroid RNA is accepted as a template for in vitro transcription by DNA-dependent DNA polymerase I and RNA polymerase from Escherichia coli

1982 ◽  
Vol 2 (11) ◽  
pp. 929-939 ◽  
Author(s):  
Wolfgang Rohde ◽  
Hans-Richard Rackwitz ◽  
Frank Boege ◽  
Heinz L. Sänger
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Dna Polymerase ◽  
Unit Length ◽  
Dna Polymerase I ◽  
Experimental Conditions ◽  
In Vitro Synthesis ◽  
Polymerase I

The RNA genome of potato spindle tuber viroid (PSTV) is transcribed in vitro into complementary DNA and RNA by DNA-dependent DNA polymerase I and RNA polymerase, respectively, from Escherichia coli. In vitro synthesis of complementary RNA produces distinct transcripts larger than unit length thus reflecting the in vivo mechanism of viroid replication. The influence of varying experimental conditions on the transcription process is studied; actinomycin D is found to drastically reduce complementary RNA synthesis from the PSTV RNA template by RNA polymerase.

scholarly journals Exonuclease III (XthA) EnforcesIn VivoDNA Cloning ofEscherichia coliTo Create Cohesive Ends

2018 ◽  
Vol 201 (5) ◽  
Author(s):  
Shingo Nozaki ◽  
Hironori Niki
Keyword(s):  
Dna Polymerase ◽  
Dna Fragments ◽  
Dna Polymerase I ◽  
Dna Cloning ◽  
Content Type ◽  
E Coli ◽  
Polymerase I ◽  
Cloning Technology

ABSTRACTEscherichia colihas an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinases but is dependent on XthA, a 3′ to 5′ exonuclease. Here,in vivocloning ofE. coliby XthA is referred to asin vivoE. colicloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced intoE. colicells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development ofin vivoDNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCECloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, anin vitrorecombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly,E. colipotentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for thisin vivocloning have realized a high level of usability, comparable to that byin vitrorecombination reactions. However, the mechanism ofin vivocloning is highly controversial. Here, we clarified the fundamental mechanism underlyingin vivocloning and also constructed a strain that was optimized forin vivocloning. Additionally, we streamlined the procedure ofin vivocloning by using a single microcentrifuge tube.

scholarly journals Reassessment of the In Vivo Functions of DNA Polymerase I and RNase H in Bacterial Cell Growth

2007 ◽  
Vol 189 (23) ◽  
pp. 8575-8583 ◽  
Author(s):  
Sanae Fukushima ◽  
Mitsuhiro Itaya ◽  
Hiroaki Kato ◽  
Naotake Ogasawara ◽  
Hirofumi Yoshikawa
Keyword(s):  
Dna Polymerase ◽  
Synthetic Lethality ◽  
Rnase H ◽  
Exonuclease Activity ◽  
Dna Polymerase I ◽  
Pol I ◽  
Exonuclease Domain ◽  
In The Wild ◽  
Polymerase I

ABSTRACT A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by rnh genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the polA and rnh genes. In a previous study, we looked for the synthetic lethality of paralogs in Bacillus subtilis and detected several essential doublet paralogs, including the polA ypcP pair. YpcP consists of only the 5′-3′ exonuclease domain. In the current study, we first confirmed that the polA genes of both Escherichia coli and B. subtilis could be completely deleted. We found that the 5′-3′ exonuclease activity encoded by either polA or ypcP xni was required for the growth of B. subtilis and E. coli. Also, the 5′-3′ exonuclease activity of Pol I was indispensable in the cyanobacterium Synechococcus elongatus. These results suggest that a 5′-3′ exonuclease activity is essential in these organisms. Our success in constructing a B. subtilis strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5′-3′ exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5′-3′ exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain.

scholarly journals RNase HIII Is Important for Okazaki Fragment Processing inBacillus subtilis

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Justin R. Randall ◽  
Taylor M. Nye ◽  
Katherine J. Wozniak ◽  
Lyle A. Simmons
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase I ◽  
Okazaki Fragment ◽  
Okazaki Fragments ◽  
Okazaki Fragment Processing ◽  
Rnase Hi ◽  
Okazaki Fragment Maturation ◽  
Polymerase I

ABSTRACTRNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strandsin vivo. The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragmentin vitrousing purified recombinant enzymes fromBacillus subtilis. We showed that RNase HII and HIII are capable of incision on Okazaki fragmentsin vitroand that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturationin vitro. Furthermore, we found that YpcP is a 5′ to 3′ nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragmentin vitro.IMPORTANCEAll cells are required to resolve the different types of RNA-DNA hybrids that formin vivo. When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processingin vitroand found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5′ to 3′ exonuclease that prefers RNA substrates with activity on Okazaki and flap substratesin vitro.

scholarly journals Exonuclease III (XthA) enforcesin vivoDNA cloning ofEscherichia colito create cohesive ends

2018 ◽  
Author(s):  
Shingo Nozaki ◽  
Hironori Niki
Keyword(s):  
Dna Polymerase ◽  
Recombination Reaction ◽  
Dna Fragments ◽  
Dna Polymerase I ◽  
Dna Cloning ◽  
E Coli ◽  
Polymerase I ◽  
Cloning Technology

AbstractEscherichia colihas an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA-cloning technology. However, the molecular mechanism by whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease. Here, in vivocloning ofE. coliby XthA is referred to as iVEC (in vivo E. colicloning). Next, we show that the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced intoE. colicells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development ofin vivoDNA-cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.ImportanceCloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently,in vitrorecombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly,E. colican take up linear double-stranded vectors, insert DNA fragments and assemble themin vivo.Thein vivocloning have realized a high level of usability comparable to that byin vitrorecombination reaction, since now it is only necessary to introduce PCR products intoE. colifor thein vivocloning. However, the mechanism ofin vivocloning is highly controversial. Here we clarified the fundamental mechanism underlyingin vivocloning of E. coli and also constructed anE. colistrain that was optimized forin vivocloning.

scholarly journals Effect of incorporation of cidofovir into DNA by human cytomegalovirus DNA polymerase on DNA elongation.

1997 ◽  
Vol 41 (3) ◽  
pp. 594-599 ◽  
Author(s):  
X Xiong ◽  
J L Smith ◽  
M S Chen
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Chain Elongation ◽  
Exonuclease Activity ◽  
Dna Chain ◽  
Alternate Substrate

Cidofovir (CDV) (HPMPC) has potent in vitro and in vivo activity against human cytomegalovirus (HCMV), CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is an inhibitor and an alternate substrate of HCMV DNA polymerase. CDV is incorporated with the correct complementation to dGMP in the template, and the incorporated CDV at the primer end is not excised by the 3'-to-5' exonuclease activity of HCMV DNA polymerase. The incorporation of a CDV molecule causes a decrease in the rate of DNA elongation for the addition of the second natural nucleotide from the singly incorporated CDV molecule. The reduction in the rate of DNA (36-mer) synthesis from an 18-mer by one incorporated CDV is 31% that of the control. However, the fidelity of HCMV DNA polymerase is maintained for the addition of the nucleotides following a single incorporated CDV molecule. The rate of DNA synthesis by HCMV DNA polymerase is drastically decreased after the incorporation of two consecutive CDV molecules; the incorporation of a third consecutive CDV molecule is not detectable. Incorporation of two CDV molecules separated by either one or two deoxynucleoside monophosphates (dAMP, dGMP, or dTMP) also drastically decreases the rate of DNA chain elongation by HCMV DNA polymerase. The rate of DNA synthesis decreases by 90% when a template which contains one internally incorporated CDV molecule is used. The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of HCMV DNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.

Sign in / Sign up

Export Citation Format

Share Document