scholarly journals Ribonucleotide incorporation characteristics around yeast autonomously replicating sequences reveal the labor division of replicative DNA polymerases

2020 ◽  
Author(s):  
Penghao Xu ◽  
Francesca Storici
Keyword(s):  
Dna Polymerase ◽  
Genome Instability ◽  
Dna Polymerases ◽  
Yeast Cells ◽  
Yeast Genome ◽  
Wild Type ◽  
Specific Sequence ◽  
Leading Strand ◽  
Mapping Techniques ◽  
Lagging Strand

ABSTRACTRibonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.

scholarly journals A mechanism for preventing asymmetric histone segregation onto replicating DNA strands

Science ◽  
2018 ◽  
Vol 361 (6409) ◽  
pp. 1386-1389 ◽  
Author(s):  
Chuanhe Yu ◽  
Haiyun Gan ◽  
Albert Serra-Cardona ◽  
Lin Zhang ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Yeast Cells ◽  
Replication Forks ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

How parental histone (H3-H4)2 tetramers, the primary carriers of epigenetic modifications, are transferred onto leading and lagging strands of DNA replication forks for epigenetic inheritance remains elusive. Here we show that parental (H3-H4)2 tetramers are assembled into nucleosomes onto both leading and lagging strands, with a slight preference for lagging strands. The lagging-strand preference increases markedly in budding yeast cells lacking Dpb3 and Dpb4, two subunits of the leading strand DNA polymerase, Pol ε, owing to the impairment of parental (H3-H4)2 transfer to leading strands. Dpb3-Dpb4 binds H3-H4 in vitro and participates in the inheritance of heterochromatin. These results indicate that different proteins facilitate the transfer of parental (H3-H4)2 onto leading versus lagging strands and that Dbp3-Dpb4 plays an important role in this poorly understood process.

scholarly journals A simple bypass assay for DNA polymerases shows hypermutating variants associated with cancer show mechanistic differences in vitro

2022 ◽  
Author(s):  
Gilles Crevel ◽  
Stephen Kearsey ◽  
Sue Cotterill
Keyword(s):  
Dna Polymerase ◽  
Natural Variation ◽  
Genome Instability ◽  
Dna Polymerases ◽  
Wild Type ◽  
Associated Diseases

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, to genome instability and its associated diseases. Recently the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants and the recognition that a subgroup of these variants have a hypermutation phenotype in tumours. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease defective and two hypermutating DNA polymerase e variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed different misincorporation characteristics to P286R, suggesting that these variants cause hypermutation by different mechanisms. Using this assay misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C to T transitions. This is consistent with the observation that C to T transitions are commonly seen in POLE mutant tumours.

Inhibition by Cyclic Guanosine 3':5'-Monophosphate of the Soluble DNA Polymerase Activity, and of Partially Purified DNA Polymerase A (DNA Polymerase I) from the Yeast Saccharomyces cerevisiae

1981 ◽  
Vol 36 (9-10) ◽  
pp. 813-819 ◽  
Author(s):  
Hans Eckstein
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nuclease Activity ◽  
Polymerase Activity ◽  
Yeast Cells ◽  
Primer Binding Site ◽  
Dna Polymerase I ◽  
Yeast Saccharomyces Cerevisiae ◽  
Main Component ◽  
Polymerase I

Abstract Dedicated to Professor Dr. Joachim Kühnau on the Occasion of His 80th Birthday cGMP, DNA Polymerase Activity, DNA Polymerase A, DNA Polymerase I, Baker's Yeast DNA polymerase activity from extracts of growing yeast cells is inhibited by cGMP. Experiments with partially purified yeast DNA polymerases show, that cGMP inhibits DNA polymerase A (DNA polymerase I from Chang), which is the main component of the soluble DNA polymerase activity in yeast extracts, by competing for the enzyme with the primer-template DNA. Since the enzyme is not only inhibited by 3',5'-cGMP, but also by 3',5'-cAMP, the 3': 5'-phosphodiester seems to be crucial for the competition between cGMP and primer. This would be inconsistent with the concept of a 3'-OH primer binding site in the enzyme. The existence of such a site in the yeast DNA polymerase A is indicated from studies with various purine nucleoside monophosphates.When various DNA polymerases are compared, inhibition by cGMP seems to be restricted to those enzymes, which are involved in DNA replication. DNA polymerases with an associated nuclease activity are not inhibited, DNA polymerase B from yeast is even activated by cGMP. Though some relations between the cGMP effect and the presumed function of the enzymes in the living cell are apparent, the biological meaning of the observations in general remains open.

scholarly journals Maintenance of Yeast Genome Integrity by RecQ Family DNA Helicases

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 205 ◽  
Author(s):  
Sonia Vidushi Gupta ◽  
Kristina Hildegard Schmidt
Keyword(s):  
Genome Instability ◽  
Dna Helicase ◽  
Model Systems ◽  
Yeast Cells ◽  
Genome Integrity ◽  
Yeast Genome ◽  
Physical Interaction ◽  
Recq Helicase ◽  
Recq Helicases ◽  
Domain Structures

With roles in DNA repair, recombination, replication and transcription, members of the RecQ DNA helicase family maintain genome integrity from bacteria to mammals. Mutations in human RecQ helicases BLM, WRN and RecQL4 cause incurable disorders characterized by genome instability, increased cancer predisposition and premature adult-onset aging. Yeast cells lacking the RecQ helicase Sgs1 share many of the cellular defects of human cells lacking BLM, including hypersensitivity to DNA damaging agents and replication stress, shortened lifespan, genome instability and mitotic hyper-recombination, making them invaluable model systems for elucidating eukaryotic RecQ helicase function. Yeast and human RecQ helicases have common DNA substrates and domain structures and share similar physical interaction partners. Here, we review the major cellular functions of the yeast RecQ helicases Sgs1 of Saccharomyces cerevisiae and Rqh1 of Schizosaccharomyces pombe and provide an outlook on some of the outstanding questions in the field.

scholarly journals Analysis of APOBEC-induced mutations in yeast strains with low levels of replicative DNA polymerases

2020 ◽  
Vol 117 (17) ◽  
pp. 9440-9450 ◽  
Author(s):  
Yang Sui ◽  
Lei Qi ◽  
Ke Zhang ◽  
Natalie Saini ◽  
Leszek J. Klimczak ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Induced Mutations ◽  
Single Stranded Dna ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Yeast Strains ◽  
Polymerase Epsilon ◽  
Low Levels ◽  
Lagging Strand

Yeast strains with low levels of the replicative DNA polymerases (alpha, delta, and epsilon) have high levels of chromosome deletions, duplications, and translocations. By examining the patterns of mutations induced in strains with low levels of DNA polymerase by the human protein APOBEC3B (a protein that deaminates cytosine in single-stranded DNA), we show dramatically elevated amounts of single-stranded DNA relative to a wild-type strain. During DNA replication, one strand (defined as the leading strand) is replicated processively by DNA polymerase epsilon and the other (the lagging strand) is replicated as short fragments initiated by DNA polymerase alpha and extended by DNA polymerase delta. In the low DNA polymerase alpha and delta strains, the APOBEC-induced mutations are concentrated on the lagging-strand template, whereas in the low DNA polymerase epsilon strain, mutations occur on the leading- and lagging-strand templates with similar frequencies. In addition, for most genes, the transcribed strand is mutagenized more frequently than the nontranscribed strand. Lastly, some of the APOBEC-induced clusters in strains with low levels of DNA polymerase alpha or delta are greater than 10 kb in length.

scholarly journals Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome

2016 ◽  
Vol 113 (21) ◽  
pp. 5916-5921 ◽  
Author(s):  
Alfredo J. Hernandez ◽  
Seung-Joo Lee ◽  
Charles C. Richardson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Fragment Length ◽  
Dna Binding Protein ◽  
Okazaki Fragment ◽  
Dna Primase ◽  
Dna Strands ◽  
Leading Strand ◽  
Rate Limiting ◽  
Lagging Strand

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

scholarly journals Human mismatch repair system corrects errors produced during lagging strand replication more effectively

2016 ◽  
Author(s):  
Maria Andrianova ◽  
Georgii A Bazykin ◽  
Sergey Nikolaev ◽  
Vladimir Seplyarskiy
Keyword(s):  
Mismatch Repair ◽  
Mutation Rates ◽  
Repair System ◽  
Wild Type ◽  
Strand Asymmetry ◽  
Exonuclease Domain ◽  
Mismatch Repair System ◽  
Dna Strands ◽  
Leading Strand ◽  
Lagging Strand

Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Different effectiveness in correction of errors produced during replication of the leading and the lagging DNA strands was reported in yeast, but this effect is poorly studied in humans. Here, we use MMR-deficient (MSI) and MMR-proficient (MSS) cancer samples to investigate properties of the human MMR. MSI, but not MSS, cancers demonstrate unequal mutation rates between the leading and the lagging strands. The direction of strand asymmetry in MSI cancers matches that observed in cancers with mutated exonuclease domain of polymerase δ, indicating that polymerase δ contributes more mutations than its leading-strand counterpart, polymerase ε. As polymerase δ primarily synthesizes DNA during the lagging strand replication, this implies that mutations produced in wild type cells during lagging strand replication are repaired by the MMR ~3 times more effectively, compared to those produced on the leading strand.

scholarly journals No support for the adaptive hypothesis of lagging-strand encoding in bacterial genomes

2020 ◽  
Author(s):  
Haoxuan Liu ◽  
Jianzhi Zhang
Keyword(s):  
Dna Replication ◽  
Comparative Genomics ◽  
Dna Polymerases ◽  
Purifying Selection ◽  
Deleterious Mutations ◽  
Bacterial Genomes ◽  
Highly Expressed Genes ◽  
Balance Hypothesis ◽  
Leading Strand ◽  
Lagging Strand

ABSTRACTGenes are preferentially encoded on the leading instead of the lagging strand of DNA replication in most bacterial genomes1. This bias likely results from selection against lagging-strand encoding, which can cause head-on collisions between DNA polymerases and RNA polymerases that induce transcriptional abortion, replication delay, and possibly mutagenesis1. But there are still genes encoded on the lagging strand, an observation that has been explained by a balance between deleterious mutations bringing genes from the leading to the lagging strand and purifying selection purging such mutations2. This mutation-selection balance hypothesis predicts that the probability that a gene is encoded on the lagging strand decreases with the detriment of its lagging-strand encoding relative to leading-strand encoding, explaining why highly expressed genes and essential genes are underrepresented on the lagging strand3,4. In a recent study, Merrikh and Merrikh proposed that the observed lagging-strand encoding is adaptive instead of detrimental, due to beneficial mutations brought by the potentially increased mutagenesis resulting from head-on collisions5. They reported empirical observations from comparative genomics that were purported to support their hypothesis5. Here we point out methodological flaws and errors in their analyses and logical problems of their interpretation. Our reanalysis of their data finds no evidence for the adaptive hypothesis.

scholarly journals Replisome bypass of transcription complexes and R-loops

2020 ◽  
Vol 48 (18) ◽  
pp. 10353-10367
Author(s):  
Jan-Gert Brüning ◽  
Kenneth J Marians
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Instability ◽  
Bacterial Dna ◽  
Template Strand ◽  
Replication Fork Progression ◽  
Leading Strand ◽  
Fork Stalling ◽  
Transcription Complexes ◽  
Lagging Strand

Abstract The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

Sign in / Sign up

Export Citation Format

Share Document