A method to select for mutator DNA polymerase δs in<i>Saccharomyces cerevisiae</i>

Murphy, Kelly; Darmawan, Hariyanto; Schultz, Amy; Silva, Elizabeth Fidalgo da; Reha-Krantz, Linda J.

doi:10.1139/g05-106

Cited by 30 publications

(22 citation statements)

References 32 publications

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“…3), and the p.Leu424 (only replaced with Met in the KF) in the B type polymerases demonstrates their importance for enzyme functionality. The side chains of p.Tyr362 and p.Leu424 together form a platform for the single-stranded primer DNA and in S. cerevisiae , the Leu479Ser (corresponding to p.Leu424) mutant completely lacks exonuclease activity (16). However, when the Leu is substituted for Ala in the T4 DNA polymerase, approximately 25% exonuclease activity is retained (17), and the severity of the potentially milder p.Leu424Val substitution is difficult to assess correctly.…”

Section: Resultsmentioning

confidence: 99%

A mutation in POLE predisposing to a multi-tumour phenotype

Rohlin

Zagoras

Nilsson

et al. 2014

International Journal of Oncology

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Somatic mutations in the POLE gene encoding the catalytic subunit of DNA polymerase ɛ have been found in sporadic colorectal cancers (CRCs) and are most likely of importance in tumour development and/or progression. Recently, families with dominantly inherited colorectal adenomas and colorectal cancer were shown to have a causative heterozygous germline mutation in the proofreading exonuclease domain of POLE. The highly penetrant mutation was associated with predisposition to CRC only and no extra-colonic tumours were observed. We have identified a mutation in a large family in which the carriers not only developed CRC, they also demonstrate a highly penetrant predisposition to extra-intestinal tumours such as ovarian, endometrial and brain tumours. The mutation, NM_006231.2:c.1089C>A, p.Asn363Lys, also located in the proofreading exonuclease domain is directly involved in DNA binding. Theoretical prediction of the amino acid substitution suggests a profound effect of the substrate binding capability and a more severe impairment of the catalytic activity compared to the previously reported germline mutation. A possible genotype to phenotype correlation for deleterious mutations in POLE might exist that needs to be considered in the follow-up of mutation carriers.

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Section: Resultsmentioning

confidence: 99%

A mutation in POLE predisposing to a multi-tumour phenotype

Rohlin

Zagoras

Nilsson

et al. 2014

International Journal of Oncology

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“…Details for the selection of mutator T4 DNA polymerases (Reha-Krantz et al, 1986; Reha-Krantz, 1988) and a similar strategy for mutator yeast DNA polymerase δ mutants (Murphy et al, 2006) are described. Several mutator T4 DNA polymerases were identified, but not the L412M-DNA polymerase because this mutant replicates DNA with relatively high fidelity as discussed ( Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

Engineering processive DNA polymerases with maximum benefit at minimum cost

2014

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DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn2+ or Ca2+. In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.

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“…We may be missing an opportunity for curative therapy, underscoring the need to improve the current systems of endometrial carcinoma risk assessment (33) 23). Through conservation studies with T4 DNA polymerase, it is likely that mutations at the residues L424P, P436R, P441L, and A456P near the exonuclease III domain also reduce proofreading resulting in a mutator phenotype (23,34,36). In Pole exonuclease-deficient mice, there is a 10-fold increase in the frequency of mutagenesis, and these mice develop tumors (37).…”

Section: Discussionmentioning

confidence: 99%

Endometrial Carcinomas with POLE Exonuclease Domain Mutations Have a Favorable Prognosis

McConechy

Talhouk

Leung

et al. 2016

Clinical Cancer Research

152

119

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Purpose: The aim of this study was to confirm the prognostic significance of POLE exonuclease domain mutations (EDM) in endometrial carcinoma patients. In addition, the effect of treatment on POLE-mutated tumors was assessed.Experimental Design: A retrospective patient cohort of 496 endometrial carcinoma patients was identified for targeted sequencing of the POLE exonuclease domain, yielding 406 evaluable tumors. Univariable and multivariable analyses were performed to determine the effect of POLE mutation status on progression-free survival (PFS), disease-specific survival (DSS), and overall survival (OS). Combining results from eight studies in a meta-analysis, we computed pooled HR for PFS, DSS, and OS.Results: POLE EDMs were identified in 39 of 406 (9.6%) endometrial carcinomas. Women with POLE-mutated endometrial carcinomas were younger, with stage I (92%) tumors, grade 3 (62%), endometrioid histology (82%), and frequent (49%) lymphovascular invasion. In univariable analysis, POLE-mutated endometrial carcinomas had significantly improved outcomes compared with patients with no EDMs for PFS, DSS, and OS. In multivariable analysis, POLE EDMs were only significantly associated with improved PFS. The effect of adjuvant treatment on POLE-mutated cases could not be determined conclusively; however, both treated and untreated patients with POLE EDMs had good outcomes. Meta-analysis revealed an association between POLE EDMs and improved PFS and DSS with pooled HRs 0.34 [95% confidence interval (CI), 0.15-0.73] and 0.35 (95% CI, 0.13-0.92), respectively.Conclusions: POLE EDMs are prognostic markers associated with excellent outcomes for endometrial carcinoma patients. Further investigation is needed to conclusively determine if treatment is necessary for this group of women.

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A method to select for mutator DNA polymerase δs inSaccharomyces cerevisiae

Cited by 30 publications

References 32 publications

A mutation in POLE predisposing to a multi-tumour phenotype

A mutation in POLE predisposing to a multi-tumour phenotype

Engineering processive DNA polymerases with maximum benefit at minimum cost

Endometrial Carcinomas with POLE Exonuclease Domain Mutations Have a Favorable Prognosis

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