Mechanism for priming DNA synthesis by yeast DNA Polymerase α

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment.

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Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

PLoS Genetics ◽

10.1371/journal.pgen.1008755 ◽

2020 ◽

Vol 16 (5) ◽

pp. e1008755 ◽

Cited By ~ 2

Author(s):

Sarina Y. Porcella ◽

Natasha C. Koussa ◽

Colin P. Tang ◽

Daphne N. Kramer ◽

Priyanka Srivastava ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Replication Origins ◽

Dna Polymerase Α ◽

Lagging Strand

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Biochemical aspects of cardiac muscle differentiation. Determination of deoxyribonucleic acid template availability and 3′-hydroxyl termini in nuclei and chromatin by using exogenous deoxyribonucleic acid polymerases

Biochemical Journal ◽

10.1042/bj1710289 ◽

1978 ◽

Vol 171 (2) ◽

pp. 289-298 ◽

Cited By ~ 9

Author(s):

William C. Claycomb

Keyword(s):

Dna Synthesis ◽

Cardiac Muscle ◽

Dna Polymerase ◽

Deoxyribonucleic Acid ◽

Dna Polymerases ◽

Dna Polymerase Α ◽

Dna Template ◽

Hydroxyl Termini ◽

Density Shift

Experiments were designed to determine whether DNA synthesis ceases in terminally differentiating cardiac muscle of the rat because the activity of the putative replicative DNA polymerase (DNA polymerase α) is lost or whether the activity of this enzyme is lost because DNA synthesis ceases. DNA-template availability and 3′-hydroxyl termini in nuclei and chromatin, isolated from cardiac muscle at various times during the developmental period in which DNA synthesis and the activity of DNA polymerase α are decreasing, were measured by using Escherichia coli DNA polymerase I, Micrococcus luteus DNA polymerase and DNA polymerase α under optimal conditions. Density-shift experiments with bromodeoxyuridine triphosphate and isopycnic analysis indicate that DNA chains being replicated semi-conservatively in vivo continue to be elongated in isolated nuclei by exogenous DNA polymerases. DNA template and 3′-hydroxyl termini available to exogenously added DNA polymerases do not change as cardiac muscle differentiates and the rate of DNA synthesis decreases and ceases in vivo. Template availability and 3′-hydroxyl termini are also not changed in nuclei isolated from cardiac muscle in which DNA synthesis had been inhibited by administration of isoproterenol and theophylline to newborn rats. DNA-template availability and 3′-hydroxyl termini, however, were substantially increased in nuclei and chromatin from cardiac muscle of adult rats. This increase is not due to elevated deoxyribonuclease activity in nuclei and chromatin of the adult. Electron microscopy indicates that this increase is also not due to dispersal of the chromatin or disruption of nuclear morphology. Density-shift experiments and isopycnic analysis of DNA from cardiac muscle of the adult show that it is more fragmented than DNA from cardiac-muscle cells that are, or have recently ceased, dividing. These studies indicate that DNA synthesis ceases in terminally differentiating cardiac muscle because the activity of a replicative DNA polymerase is lost, rather than the activity of this enzyme being lost because DNA synthesis ceases.

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DNA Synthesis on a Double-stranded DNA Template by the T4 Bacteriophage DNA Polymerase and the T4 Gene 32 DNA Unwinding Protein

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)79779-6 ◽

1974 ◽

Vol 249 (17) ◽

pp. 5668-5676

Author(s):

Nancy G. Nossal

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Dna Unwinding ◽

Double Stranded Dna ◽

T4 Bacteriophage ◽

Gene 32 ◽

Dna Template

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The Function of DNA Polymerase α at Telomeric G Tails Is Important for Telomere Homeostasis

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.786-796.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 786-796 ◽

Cited By ~ 66

Author(s):

Aegina Adams Martin ◽

Isabelle Dionne ◽

Raymund J. Wellinger ◽

Connie Holm

Keyword(s):

Telomere Length ◽

Dna Polymerase ◽

Telomere Maintenance ◽

Replication Machinery ◽

Telomeric Dna ◽

Dna Polymerase Α ◽

Telomeric Silencing ◽

Pass Through ◽

Telomere Lengthening ◽

Lagging Strand

ABSTRACT Telomere length control is influenced by several factors, including telomerase, the components of telomeric chromatin structure, and the conventional replication machinery. Although known components of the replication machinery can influence telomere length equilibrium, little is known about why mutations in certain replication proteins cause dramatic telomere lengthening. To investigate the cause of telomere elongation in cdc17/pol1 (DNA polymerase α) mutants, we examined telomeric chromatin, as measured by its ability to repress transcription on telomere-proximal genes, and telomeric DNA end structures in pol1-17 mutants. pol1-17 mutants with elongated telomeres show a dramatic loss of the repression of telomere-proximal genes, or telomeric silencing. In addition,cdc17/pol1 mutants grown under telomere-elongating conditions exhibit significant increases in single-stranded character in telomeric DNA but not at internal sequences. The single strandedness is manifested as a terminal extension of the G-rich strand (G tails) that can occur independently of telomerase, suggesting thatcdc17/pol1 mutants exhibit defects in telomeric lagging-strand synthesis. Interestingly, the loss of telomeric silencing and the increase in the sizes of the G tails at the telomeres temporally coincide and occur before any detectable telomere lengthening is observed. Moreover, the G tails observed incdc17/pol1 mutants incubated at the semipermissive temperature appear only when the cells pass through S phase and are processed by the time cells reach G1. These results suggest that lagging-strand synthesis is coordinated with telomerase-mediated telomere maintenance to ensure proper telomere length control.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1604894113 ◽

2016 ◽

Vol 113 (21) ◽

pp. 5916-5921 ◽

Cited By ~ 8

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.

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