scholarly journals High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jessica S. Williams ◽  
Percy P. Tumbale ◽  
Mercedes E. Arana ◽  
Julian A. Rana ◽  
R. Scott Williams ◽  
...  
Keyword(s):  
Dna Replication ◽  
Metal Binding ◽  
Nuclear Dna ◽  
Nuclear Genome ◽  
Structural Data ◽  
High Fidelity ◽  
Okazaki Fragment ◽  
Dna Ligation ◽  
Okazaki Fragment Maturation ◽  
Insertion Mutations

AbstractDNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

scholarly journals The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

2017 ◽  
Vol 199 (13) ◽  
Author(s):  
Brett W. Burkhart ◽  
Lubomira Cubonova ◽  
Margaret R. Heider ◽  
Zvi Kelman ◽  
John N. Reeve ◽  
...  
Keyword(s):  
Dna Replication ◽  
Okazaki Fragment ◽  
Content Type ◽  
Thermococcus Kodakarensis ◽  
Okazaki Fragments ◽  
Domains Of Life ◽  
Okazaki Fragment Maturation ◽  
Polymerase I ◽  
Lagging Strand

ABSTRACT Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5′-to-3′ exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo. RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase. IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea. Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

scholarly journals Low Nucleotide Diversity in Chimpanzees and Bonobos

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1511-1518 ◽  
Author(s):  
Ning Yu ◽  
Michael I Jensen-Seaman ◽  
Leona Chemnick ◽  
Judith R Kidd ◽  
Amos S Deinard ◽  
...  
Keyword(s):  
Nucleotide Diversity ◽  
Nuclear Dna ◽  
Demographic History ◽  
Nuclear Genome ◽  
Limited Data ◽  
Effective Population ◽  
East Central ◽  
Dna Diversity ◽  
History Of ◽  
Mtdna Diversity

Abstract Comparison of the levels of nucleotide diversity in humans and apes may provide much insight into the mechanisms of maintenance of DNA polymorphism and the demographic history of these organisms. In the past, abundant mitochondrial DNA (mtDNA) polymorphism data indicated that nucleotide diversity (π) is more than threefold higher in chimpanzees than in humans. Furthermore, it has recently been claimed, on the basis of limited data, that this is also true for nuclear DNA. In this study we sequenced 50 noncoding, nonrepetitive DNA segments randomly chosen from the nuclear genome in 9 bonobos and 17 chimpanzees. Surprisingly, the π value for bonobos is only 0.078%, even somewhat lower than that (0.088%) for humans for the same 50 segments. The π values are 0.092, 0.130, and 0.082% for East, Central, and West African chimpanzees, respectively, and 0.132% for all chimpanzees. These values are similar to or at most only 1.5 times higher than that for humans. The much larger difference in mtDNA diversity than in nuclear DNA diversity between humans and chimpanzees is puzzling. We speculate that it is due mainly to a reduction in effective population size (Ne) in the human lineage after the human-chimpanzee divergence, because a reduction in Ne has a stronger effect on mtDNA diversity than on nuclear DNA diversity.

scholarly journals Risk of cardiac manifestations in adult mitochondrial disease caused by nuclear genetic defects

Open Heart ◽  
2021 ◽  
Vol 8 (1) ◽  
pp. e001510
Author(s):  
Albert Zishen Lim ◽  
Daniel M Jones ◽  
Matthew G D Bates ◽  
Andrew M Schaefer ◽  
John O'Sullivan ◽  
...  
Keyword(s):  
Mitochondrial Disease ◽  
Nuclear Dna ◽  
Laboratory Data ◽  
Nuclear Genome ◽  
Nuclear Gene ◽  
Left Ventricular ◽  
Adult Patients ◽  
Limited Information ◽  
Cardiac Abnormalities ◽  
Cardiac Manifestations

ObjectiveRegular cardiac surveillance is advocated for patients with primary mitochondrial DNA disease. However, there is limited information to guide clinical practice in mitochondrial conditions caused by nuclear DNA defects. We sought to determine the frequency and spectrum of cardiac abnormalities identified in adult mitochondrial disease originated from the nuclear genome.MethodsAdult patients with a genetically confirmed mitochondrial disease were identified and followed up at the national clinical service for mitochondrial disease in Newcastle upon Tyne, UK (January 2009 to December 2018). Case notes, molecular genetics reports, laboratory data and cardiac investigations, including serial electrocardiograms and echocardiograms, were reviewed.ResultsIn this cohort-based observational study, we included 146 adult patients (92 women) (mean age 53.6±18.7 years, 95% CI 50.6 to 56.7) with a mean follow-up duration of 7.9±5.1 years (95% CI 7.0 to 8.8). Eleven different nuclear genotypes were identified: TWNK, POLG, RRM2B, OPA1, GFER, YARS2, TYMP, ETFDH, SDHA, TRIT1 and AGK. Cardiac abnormalities were detected in 14 patients (9.6%). Seven of these patients (4.8%) had early-onset cardiac manifestations: hypertrophic cardiomyopathy required cardiac transplantation (AGK; n=2/2), left ventricular (LV) hypertrophy and bifascicular heart block (GFER; n=2/3) and mild LV dysfunction (GFER; n=1/3, YARS2; n=1/2, TWNK; n=1/41). The remaining seven patients had acquired heart disease most likely related to conventional cardiovascular risk factors and presented later in life (14.6±12.8 vs 55.1±8.9 years, p<0.0001).ConclusionsOur findings demonstrate that the risk of cardiac involvement is genotype specific, suggesting that routine cardiac screening is not indicated for most adult patients with nuclear gene-related mitochondrial disease.

scholarly journals Mitochondrial DNA Methylation and Human Diseases

2021 ◽  
Vol 22 (9) ◽  
pp. 4594
Author(s):  
Andrea Stoccoro ◽  
Fabio Coppedè
Keyword(s):  
Dna Methylation ◽  
Mitochondrial Dna ◽  
Mitochondrial Genome ◽  
Nuclear Dna ◽  
Epigenetic Modification ◽  
Nuclear Genome ◽  
Epigenetic Modifications ◽  
Human Diseases ◽  
Loop Region ◽  
D Loop

Epigenetic modifications of the nuclear genome, including DNA methylation, histone modifications and non-coding RNA post-transcriptional regulation, are increasingly being involved in the pathogenesis of several human diseases. Recent evidence suggests that also epigenetic modifications of the mitochondrial genome could contribute to the etiology of human diseases. In particular, altered methylation and hydroxymethylation levels of mitochondrial DNA (mtDNA) have been found in animal models and in human tissues from patients affected by cancer, obesity, diabetes and cardiovascular and neurodegenerative diseases. Moreover, environmental factors, as well as nuclear DNA genetic variants, have been found to impair mtDNA methylation patterns. Some authors failed to find DNA methylation marks in the mitochondrial genome, suggesting that it is unlikely that this epigenetic modification plays any role in the control of the mitochondrial function. On the other hand, several other studies successfully identified the presence of mtDNA methylation, particularly in the mitochondrial displacement loop (D-loop) region, relating it to changes in both mtDNA gene transcription and mitochondrial replication. Overall, investigations performed until now suggest that methylation and hydroxymethylation marks are present in the mtDNA genome, albeit at lower levels compared to those detectable in nuclear DNA, potentially contributing to the mitochondria impairment underlying several human diseases.

scholarly journals Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis

2008 ◽  
Vol 283 (49) ◽  
pp. 34129-34140 ◽  
Author(s):  
Carrie M. Stith ◽  
Joan Sterling ◽  
Michael A. Resnick ◽  
Dmitry A. Gordenin ◽  
Peter M. Burgers
Keyword(s):  
Strand Displacement ◽  
Okazaki Fragment ◽  
Okazaki Fragment Maturation

scholarly journals The TREX2 3′→ 5′ Exonuclease Physically Interacts with DNA Polymerase δ and Increases Its Accuracy

2002 ◽  
Vol 2 ◽  
pp. 275-281 ◽  
Author(s):  
Igor V. Shevelev ◽  
Kristijan Ramadan ◽  
Ulrich Hubscher
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Error Rates ◽  
High Fidelity ◽  
Replication Machinery ◽  
Dna Polymerase Δ ◽  
Pol I ◽  
Calf Thymus ◽  
Mutation Assay ◽  
Forward Mutation

Proofreading function by the 3′→ 5′ exonuclease of DNA polymerase δ (pol δ) is consistent with the observation that deficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol δdfrom isotonic (pol δi) and detergent (pol δd) calf thymus extracts. Pol δdhad a 20-fold higher ratio of exonuclease to DNA polymerase than pol δi. This was due to the physical association of the TREX2 exonuclease to pol δd, which was missing from pol δi. Pol δdwas fivefold more accurate than pol δiunder error-prone conditions (1 μM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 μM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3′→ 5′ exonucleases, such as TREX2, through its association with pol I can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Sign in / Sign up

Export Citation Format

Share Document