scholarly journals Initiating DNA replication: a matter of prime importance

2019 ◽  
Vol 47 (1) ◽  
pp. 351-356 ◽  
Author(s):  
Stephen D. Bell
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
De Novo ◽  
Broad Class ◽  
Genome Replication ◽  
Extrachromosomal Element ◽  
Replication Systems ◽  
Dna Chain ◽  
Mechanistic Basis ◽  
Cellular Dna

Abstract It has been known for decades that the principal replicative DNA polymerases that effect genome replication are incapable of starting DNA synthesis de novo. Rather, they require a 3′-OH group from which to extend a DNA chain. Cellular DNA replication systems exploit a dedicated, limited processivity RNA polymerase, termed primase, that synthesizes a short oligoribonucleotide primer which is then extended by a DNA polymerase. Thus, primases can initiate synthesis, proceed with primer elongation for a short distance then transfer the primer to a DNA polymerase. Despite these well-established properties, the mechanistic basis of these dynamic behaviours has only recently been established. In the following, the author will describe recent insights from studies of the related eukaryotic and archaeal DNA primases. Significantly, the general conclusions from these studies likely extend to a broad class of extrachromosomal element-associated primases as well as the human primase-related DNA repair enzyme, PrimPol.

scholarly journals DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1739
Author(s):  
Chen-Yu Lo ◽  
Yang Gao
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Molecular Mechanisms ◽  
Bacteriophage T4 ◽  
Dna Helicase ◽  
Model Systems ◽  
Genome Replication ◽  
Template Strand ◽  
Replication Systems ◽  
Mechanistic Basis

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.

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.

Mammalian DNA polymerase α : a replication-competent holoenzyme form from calf thymus

1987 ◽  
Vol 317 (1187) ◽  
pp. 421-428 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Consensus Sequence ◽  
Porcine Circovirus ◽  
Chromosomal Replication ◽  
Dna Polymerase Α ◽  
Calf Thymus ◽  
Dna Replication Origins ◽  
Cellular Dna

Calf thymus DNA polymerase α, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli , can be isolated as a distinct complex. A specific multiprotein form of the polymerase α, a form designated replication-com petent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme α selectively initiates pcv DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.

scholarly journals Archaeal DNA polymerases: new frontiers in DNA replication and repair

2018 ◽  
Vol 2 (4) ◽  
pp. 503-516 ◽  
Author(s):  
Christopher D.O. Cooper
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Amplification ◽  
Model Systems ◽  
Genome Replication ◽  
Sequencing Technologies ◽  
Dna Replication And Repair ◽  
Polymerase Chain

Archaeal DNA polymerases have long been studied due to their superior properties for DNA amplification in the polymerase chain reaction and DNA sequencing technologies. However, a full comprehension of their functions, recruitment and regulation as part of the replisome during genome replication and DNA repair lags behind well-established bacterial and eukaryotic model systems. The archaea are evolutionarily very broad, but many studies in the major model systems of both Crenarchaeota and Euryarchaeota are starting to yield significant increases in understanding of the functions of DNA polymerases in the respective phyla. Recent advances in biochemical approaches and in archaeal genetic models allowing knockout and epitope tagging have led to significant increases in our understanding, including DNA polymerase roles in Okazaki fragment maturation on the lagging strand, towards reconstitution of the replisome itself. Furthermore, poorly characterised DNA polymerase paralogues are finding roles in DNA repair and CRISPR immunity. This review attempts to provide a current update on the roles of archaeal DNA polymerases in both DNA replication and repair, addressing significant questions that remain for this field.

scholarly journals Mechanistic basis for microhomology identification and genome scarring by polymerase theta

2020 ◽  
Vol 117 (15) ◽  
pp. 8476-8485 ◽  
Author(s):  
Juan Carvajal-Garcia ◽  
Jang-Eun Cho ◽  
Pablo Carvajal-Garcia ◽  
Wanjuan Feng ◽  
Richard D. Wood ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
De Novo ◽  
Genome Instability ◽  
End Joining ◽  
Complementary Sequence ◽  
Chromosome Breaks ◽  
Complete Repair ◽  
Cancer Genomes ◽  
Mechanistic Basis ◽  
Scanning Mechanism

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

scholarly journals Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta

2019 ◽  
Author(s):  
Juan Carvajal-Garcia ◽  
Jang-Eun Cho ◽  
Pablo Carvajal-Garcia ◽  
Wanjuan Feng ◽  
Richard D. Wood ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
De Novo ◽  
Genome Instability ◽  
End Joining ◽  
Complementary Sequence ◽  
Chromosome Breaks ◽  
Complete Repair ◽  
Cancer Genomes ◽  
Mechanistic Basis ◽  
Scanning Mechanism

AbstractDNA Polymerase Theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3’ single stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3’ terminus and favoring bi-directional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3-30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification, and shows both how it mitigates limitations implicit in the microhomology requirement, and generates distinctive genomic scars associated with pathogenic genome instability.

scholarly journals DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

1996 ◽  
Vol 15 (9) ◽  
pp. 2298-2305 ◽  
Author(s):  
T. Zlotkin ◽  
G. Kaufmann ◽  
Y. Jiang ◽  
M. Y. Lee ◽  
L. Uitto ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Polymerase Epsilon ◽  
Polymerase Epsilon ◽  
Cellular Dna

scholarly journals Two Immunologically Distinct Human DNA Polymerase α-Primase Subpopulations Are Involved in Cellular DNA Replication

2001 ◽  
Vol 21 (7) ◽  
pp. 2581-2593 ◽  
Author(s):  
Silke Dehde ◽  
Gabor Rohaly ◽  
Oliver Schub ◽  
Heinz-Peter Nasheuer ◽  
Wolfgang Bohn ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Brdu Incorporation ◽  
Metabolic Labeling ◽  
Dna Polymerase Α ◽  
Origin Binding Protein ◽  
Cellular Dna

ABSTRACT Metabolic labeling of primate cells revealed the existence of phosphorylated and hypophosphorylated DNA polymerase α-primase (Pol-Prim) populations that are distinguishable by monoclonal antibodies. Cell cycle studies showed that the hypophosphorylated form was found in a complex with PP2A and cyclin E-Cdk2 in G1, whereas the phosphorylated enzyme was associated with a cyclin A kinase in S and G2. Modification of Pol-Prim by PP2A and Cdks regulated the interaction with the simian virus 40 origin-binding protein large T antigen and thus initiation of DNA replication. Confocal microscopy demonstrated nuclear colocalization of hypophosphorylated Pol-Prim with MCM2 in S phase nuclei, but its presence preceded 5-bromo-2′-deoxyuridine (BrdU) incorporation. The phosphorylated replicase exclusively colocalized with the BrdU signal, but not with MCM2. Immunoprecipitation experiments proved that only hypophosphorylated Pol-Prim associated with MCM2. The data indicate that the hypophosphorylated enzyme initiates DNA replication at origins, and the phosphorylated form synthesizes the primers for the lagging strand of the replication fork.

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