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.

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 Mutational analysis of the mRNA operator for T4 DNA polymerase.

Genetics ◽  
1991 ◽  
Vol 128 (2) ◽  
pp. 203-213 ◽  
Author(s):  
M D Andrake ◽  
J D Karam
Keyword(s):  
Dna Polymerase ◽  
Mutational Analysis ◽  
Bacteriophage T4 ◽  
Spatial Arrangement ◽  
Loop Sequence ◽  
Shine Dalgarno Sequence ◽  
In Vitro Effects ◽  
Rna Hairpin

Abstract Biosynthesis of bacteriophage T4 DNA polymerase is autogenously regulated at the translational level. The enzyme, product of gene 43, represses its own translation by binding to its mRNA 5' to the initiator AUG at a 36-40 nucleotide segment that includes the Shine-Dalgarno sequence and a putative RNA hairpin structure consisting of a 5-base-pair stem and an 8-base loop. We constructed mutations that either disrupted the stem or altered specific loop residues of the hairpin and found that many of these mutations, including single-base changes in the loop sequence, diminished binding of purified T4 DNA polymerase to its RNA in vitro (as measured by a gel retardation assay) and derepressed synthesis of the enzyme in vivo (as measured in T4 infections and by recombinant-plasmid-mediated expression). In vitro effects, however, were not always congruent with in vivo effects. For example, stem pairing with a sequence other than wild-type resulted in normal protein binding in vitro but derepression of protein synthesis in vivo. Similarly, a C----A change in the loop had a small effect in vitro and a strong effect in vivo. In contrast, an A----U change near the base of the hairpin that was predicted to increase the length of the base-paired stem had small effects both in vitro and in vivo. The results suggest that interaction of T4 DNA polymerase with its structured RNA operator depends on the spatial arrangement of specific nucleotide residues and is subject to modulation in vivo.

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.

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 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 DNA polymerase II of Escherichia coli in the bypass of abasic sites in vivo.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 439-448 ◽  
Author(s):  
I Tessman ◽  
M A Kennedy
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Quantitative Difference ◽  
Spontaneous Mutations ◽  
Pol Ii ◽  
Abasic Sites ◽  
Dna Polymerase Ii

Abstract The function of DNA polymerase II of Escherichia coli is an old question. Any phenotypic character that Pol II may confer upon the cell has escaped detection since the polymerase was discovered 24 yr ago. Although it has been shown that Pol II enables DNA synthesis to proceed past abasic sites in vitro, no role is known for it in the bypass of those lesions in vivo. From a study of phage S13 single-stranded DNA, we now report SOS conditions under which Pol II is needed for DNA synthesis to proceed past abasic sites with 100% efficiency in vivo. Overproduction of the GroES+L+ heat shock proteins, which are members of a ubiquitous family of molecular chaperones, eliminated this requirement for Pol II, which may explain why the role of Pol II in SOS repair had eluded discovery. Mutagenesis accompanied SOS bypass of abasic sites when the original occupant had been cytosine but not when it had been thymine; the quantitative difference is shown to imply that adenine was inserted opposite the abasic sites at least 99.7% of the time, which is an especially strict application of the A-rule. Most, but not all, spontaneous mutations from Rifs to Rifr, whether in a recA+ or a recA(Prtc) cell, require Pol II; while this suggests that cryptic abasic lesions are a likely source of spontaneous mutations, it also shows that such lesions cannot be the exclusive source.

scholarly journals The Werner Syndrome Exonuclease Facilitates DNA Degradation and High Fidelity DNA Polymerization by Human DNA Polymerase δ

2012 ◽  
Vol 287 (15) ◽  
pp. 12480-12490 ◽  
Author(s):  
Ashwini S. Kamath-Loeb ◽  
Jiang-Cheng Shen ◽  
Michael W. Schmitt ◽  
Lawrence A. Loeb
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Genome Stability ◽  
Werner Syndrome ◽  
Premature Aging ◽  
High Fidelity ◽  
Dna Polymerase Δ ◽  
Family Loss

DNA Polymerase δ (Pol δ) and the Werner syndrome protein, WRN, are involved in maintaining cellular genomic stability. Pol δ synthesizes the lagging strand during replication of genomic DNA and also functions in the synthesis steps of DNA repair and recombination. WRN is a member of the RecQ helicase family, loss of which results in the premature aging and cancer-prone disorder, Werner syndrome. Both Pol δ and WRN encode 3′ → 5′ DNA exonuclease activities. Pol δ exonuclease removes 3′-terminal mismatched nucleotides incorporated during replication to ensure high fidelity DNA synthesis. WRN exonuclease degrades DNA containing alternate secondary structures to prevent formation and enable resolution of stalled replication forks. We now observe that similarly to WRN, Pol δ degrades alternate DNA structures including bubbles, four-way junctions, and D-loops. Moreover, WRN and Pol δ form a complex with enhanced ability to hydrolyze these structures. We also present evidence that WRN can proofread for Pol δ; WRN excises 3′-terminal mismatches to enable primer extension by Pol δ. Consistent with ourin vitroobservations, we show that WRN contributes to the maintenance of DNA synthesis fidelityin vivo. Cells expressing limiting amounts (∼10% of normal) of WRN have elevated mutation frequencies compared with wild-type cells. Together, our data highlight the importance of WRN exonuclease activity and its cooperativity with Pol δ in preserving genome stability, which is compromised by the loss of WRN in Werner syndrome.

scholarly journals Dissecting the Fidelity of Bacteriophage RB69 DNA Polymerase: Site-Specific Modulation of Fidelity by Polymerase Accessory Proteins

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1003-1018 ◽  
Author(s):  
Anna Bebenek ◽  
Geraldine T Carver ◽  
Holly Kloos Dressman ◽  
Farid A Kadyrov ◽  
Joseph K Haseman ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Important Determinant ◽  
Mutation Rates ◽  
Accessory Proteins ◽  
Wild Type ◽  
Mutational Spectra ◽  
Base Selection ◽  
Bacteriophage Rb69

Abstract Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3′ exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol+ Exo+) enzyme, an exonuclease-deficient mutator variant (Pol+ Exo-), mutator variants with substitutions at Tyr567 in the polymerase active site (PolM Exo+), and the double mutator PolM Exo-. Comparing the mutational spectra of the Pol+ Exo- and Pol+ Exo+ enzymes revealed the patterns and efficiencies of proofreading, while Tyr567 was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.

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