scholarly journals Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase μ

2001 ◽  
Vol 21 (23) ◽  
pp. 7995-8006 ◽  
Author(s):  
Yanbin Zhang ◽  
Xiaohua Wu ◽  
Fenghua Yuan ◽  
Zhongwen Xie ◽  
Zhigang Wang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Somatic Hypermutation ◽  
Biochemical Properties ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Synthesis Mechanism ◽  
Biochemical Analyses

ABSTRACT DNA polymerase μ (Polμ) is a newly identified member of the polymerase X family. The biological function of Polμ is not known, although it has been speculated that human Polμ may be a somatic hypermutation polymerase. To help understand the in vivo function of human Polμ, we have performed in vitro biochemical analyses of the purified polymerase. Unlike any other DNA polymerases studied thus far, human Polμ catalyzed frameshift DNA synthesis with an unprecedentedly high frequency. In the sequence contexts examined, −1 deletion occurred as the predominant DNA synthesis mechanism opposite the single-nucleotide repeat sequences AA, GG, TT, and CC in the template. Thus, the fidelity of DNA synthesis by human Polμ was largely dictated by the sequence context. Human Polμ was able to efficiently extend mismatched bases mainly by a frameshift synthesis mechanism. With the primer ends, containing up to four mismatches, examined, human Polμ effectively realigned the primer to achieve annealing with a microhomology region in the template several nucleotides downstream. As a result, human Polμ promoted microhomology search and microhomology pairing between the primer and the template strands of DNA. These results show that human Polμ is much more prone to cause frameshift mutations than base substitutions. The biochemical properties of human Polμ suggest a function in nonhomologous end joining and V(D)J recombination through its microhomology searching and pairing activities but do not support a function in somatic hypermutation.

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.

scholarly journals The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

2008 ◽  
Vol 29 (5) ◽  
pp. 1116-1122 ◽  
Author(s):  
Laurent Malivert ◽  
Isabelle Callebaut ◽  
Paola Rivera-Munoz ◽  
Alain Fischer ◽  
Jean-Paul Mornon ◽  
...  
Keyword(s):  
Dna Repair ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
Repair Pathway ◽  
End Joining ◽  
Functional Assays ◽  
The Core ◽  
Ligase Iv

ABSTRACT The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

Nonhomologous recombination in human cells

1994 ◽  
Vol 14 (1) ◽  
pp. 156-169
Author(s):  
M K Derbyshire ◽  
L H Epstein ◽  
C S Young ◽  
P L Munz ◽  
R Fishel
Keyword(s):  
Human Cells ◽  
Nonhomologous End Joining ◽  
Dna Fragments ◽  
End Joining ◽  
Major Pathway ◽  
Biochemical Basis ◽  
Nonhomologous Recombination ◽  
Mammalian Cell Lines

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

scholarly journals Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

2004 ◽  
Vol 24 (7) ◽  
pp. 2734-2746 ◽  
Author(s):  
Atsuko Niimi ◽  
Siripan Limsirichaikul ◽  
Shonen Yoshida ◽  
Shigenori Iwai ◽  
Chikahide Masutani ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Pyrimidine Dimer ◽  
Replication Fidelity ◽  
Dna Polymerase Α ◽  
Replication Errors ◽  
Translesion Dna

ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

scholarly journals Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

2015 ◽  
Vol 112 (20) ◽  
pp. E2575-E2584 ◽  
Author(s):  
Dylan A. Reid ◽  
Sarah Keegan ◽  
Alejandra Leo-Macias ◽  
Go Watanabe ◽  
Natasha T. Strande ◽  
...  
Keyword(s):  
Single Molecule ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv ◽  
End To End

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

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.

DNA Polymerase Fidelity: From Genetics Toward a Biochemical Understanding

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1475-1482
Author(s):  
Myron F Goodman ◽  
D Kuchnir Fygenson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Theoretical Models ◽  
Model Systems ◽  
Polymerase Fidelity ◽  
Base Selection ◽  
Physical Biochemistry

Abstract This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful—first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered. For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.

Tokishakuyakusan Effect on DNA Polymerase α Activity in Relationship to DNA Synthesis before and/or after the LH/FSH Surge in Rats

1995 ◽  
Vol 23 (03n04) ◽  
pp. 231-242 ◽  
Author(s):  
Satoshi Usuki ◽  
Eri Kotani ◽  
Yumiko Kawakura ◽  
Masaki Sano ◽  
Yukitaka Katsura ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Estrous Cycle ◽  
Dna Polymerase ◽  
Preovulatory Follicle ◽  
Dna Polymerase Β ◽  
Dna Polymerase Α ◽  
Immature Rats ◽  
Α Activity

DNA polymerase α activity in ovaries of mature cycling rats during the normal estrous cycle changed in a cyclic manner with a peak at 1800 h in proestrus. Tokishakuyakusan (TS) in vivo did not affect the changes in DNA polymerase α and β activities during the estrous cycle. LH and FSH at 1000 or 1700 h in proestrus increased DNA polymerase α activity, but the DNA polymerase α activity induced by LH or FSH was not significantly affected by the addition of TS. DNA polymerase β activity did not change with LH, FSH or TS. In PMS-treated or -untreated immature rats, TS enhanced ovarian DNA polymerase α activity but had no significant effect on LH or FSH action. In ovaries, incubated in vitro, in untreated mature or immature rats, TS enhanced ovarian DNA polymerase α activity but had no significant effect on LH or FSH action. These results suggest that TS stimulates ovarian DNA polymerase α activity in relationship to DNA synthesis and does not affect the effect of LH or FSH on the activity by preovulatory follicle before and/or after the LH/FSH surge.

scholarly journals BRCA1 ensures genome integrity by eliminating estrogen-induced pathological topoisomerase II–DNA complexes

2018 ◽  
Vol 115 (45) ◽  
pp. E10642-E10651 ◽  
Author(s):  
Hiroyuki Sasanuma ◽  
Masataka Tsuda ◽  
Suguru Morimoto ◽  
Liton Kumar Saha ◽  
Md Maminur Rahman ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Topoisomerase Ii ◽  
Germ Line ◽  
Critical Role ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Er Positive

Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5′ ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5′ TOP2 adducts in G1phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G1phase. We disrupted theBRCA1gene in53BP1-deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G1phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining.BRCA1-deficient cells showed a decrease in etoposide-induced MRE11 foci in G1phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G1-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.

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