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Annual Review of Biochemistry Jun 2017This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair... (Review)
Review
This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.
Topics: Animals; Cell Cycle Proteins; DNA; DNA Helicases; DNA Polymerase I; DNA Polymerase II; DNA Polymerase III; DNA Replication; DNA-Binding Proteins; Eukaryotic Cells; Humans; Minichromosome Maintenance Proteins
PubMed: 28301743
DOI: 10.1146/annurev-biochem-061516-044709 -
Viruses Oct 2021DNA replication is an integral step in the herpes simplex virus type 1 (HSV-1) life cycle that is coordinated with the cellular DNA damage response, repair and... (Review)
Review
DNA replication is an integral step in the herpes simplex virus type 1 (HSV-1) life cycle that is coordinated with the cellular DNA damage response, repair and recombination of the viral genome, and viral gene transcription. HSV-1 encodes its own DNA replication machinery, including an origin binding protein (UL9), single-stranded DNA binding protein (ICP8), DNA polymerase (UL30), processivity factor (UL42), and a helicase/primase complex (UL5/UL8/UL52). In addition, HSV-1 utilizes a combination of accessory viral and cellular factors to coordinate viral DNA replication with other viral and cellular processes. The purpose of this review is to outline the roles of viral and cellular proteins in HSV-1 DNA replication and replication-coupled processes, and to highlight how HSV-1 may modify and adapt cellular proteins to facilitate productive infection.
Topics: DNA Helicases; DNA Primase; DNA Replication; DNA, Viral; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Genome, Viral; Herpesvirus 1, Human; Humans; Viral Proteins; Virus Replication
PubMed: 34696446
DOI: 10.3390/v13102015 -
Nature Jun 2022Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase...
Chromosome replication is performed by a complex and intricate ensemble of proteins termed the replisome, where the DNA polymerases Polδ and Polε, DNA polymerase α-primase (Polα) and accessory proteins including AND-1, CLASPIN and TIMELESS-TIPIN (respectively known as Ctf4, Mrc1 and Tof1-Csm3 in Saccharomyces cerevisiae) are organized around the CDC45-MCM-GINS (CMG) replicative helicase. Because a functional human replisome has not been reconstituted from purified proteins, how these factors contribute to human DNA replication and whether additional proteins are required for optimal DNA synthesis are poorly understood. Here we report the biochemical reconstitution of human replisomes that perform fast and efficient DNA replication using 11 purified human replication factors made from 43 polypeptides. Polε, but not Polδ, is crucial for optimal leading-strand synthesis. Unexpectedly, Polε-mediated leading-strand replication is highly dependent on the sliding-clamp processivity factor PCNA and the alternative clamp loader complex CTF18-RFC. We show how CLASPIN and TIMELESS-TIPIN contribute to replisome progression and demonstrate that, in contrast to the budding yeast replisome, AND-1 directly augments leading-strand replication. Moreover, although AND-1 binds to Polα, the interaction is dispensable for lagging-strand replication, indicating that Polα is functionally recruited via an AND-1-independent mechanism for priming in the human replisome. Collectively, our work reveals how the human replisome achieves fast and efficient leading-strand and lagging-strand DNA replication, and provides a powerful system for future studies of the human replisome and its interactions with other DNA metabolic processes.
Topics: DNA; DNA Helicases; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Humans; Multienzyme Complexes; Time Factors
PubMed: 35585232
DOI: 10.1038/s41586-022-04759-1 -
Annual Review of Microbiology Sep 2020It is now well recognized that the information processing machineries of archaea are far more closely related to those of eukaryotes than to those of their prokaryotic... (Review)
Review
It is now well recognized that the information processing machineries of archaea are far more closely related to those of eukaryotes than to those of their prokaryotic cousins, the bacteria. Extensive studies have been performed on the structure and function of the archaeal DNA replication origins, the proteins that define them, and the macromolecular assemblies that drive DNA unwinding and nascent strand synthesis. The results from various archaeal organisms across the archaeal domain of life show surprising levels of diversity at many levels-ranging from cell cycle organization to chromosome ploidy to replication mode and nature of the replicative polymerases. In the following, we describe recent advances in the field, highlighting conserved features and lineage-specific innovations.
Topics: Archaea; Archaeal Proteins; DNA Replication; DNA, Archaeal; Models, Molecular; Protein Binding
PubMed: 32503372
DOI: 10.1146/annurev-micro-020518-115443 -
Cell Reports May 2022The MDM2 oncoprotein antagonizes the tumor suppressor p53 by physical interaction and ubiquitination. However, it also sustains the progression of DNA replication forks,...
The MDM2 oncoprotein antagonizes the tumor suppressor p53 by physical interaction and ubiquitination. However, it also sustains the progression of DNA replication forks, even in the absence of functional p53. Here, we show that MDM2 binds, inhibits, ubiquitinates, and destabilizes poly(ADP-ribose) polymerase 1 (PARP1). When cellular MDM2 levels are increased, this leads to accelerated progression of DNA replication forks, much like pharmacological inhibition of PARP1. Conversely, overexpressed PARP1 restores normal fork progression despite elevated MDM2. Strikingly, MDM2 profoundly reduces the frequency of fork reversal, revealed as four-way junctions through electron microscopy. Depletion of RECQ1 or the primase/polymerase (PRIMPOL) reverses the MDM2-mediated acceleration of the nascent DNA elongation rate. MDM2 also increases the occurrence of micronuclei, and it exacerbates camptothecin-induced cell death. In conclusion, high MDM2 levels phenocopy PARP inhibition in modulation of fork restart, representing a potential vulnerability of cancer cells.
Topics: DNA; DNA Damage; DNA Primase; DNA Replication; Tumor Suppressor Protein p53
PubMed: 35649362
DOI: 10.1016/j.celrep.2022.110879 -
Molecular Cell Oct 2021BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and...
BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.
Topics: Animals; Antineoplastic Agents; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; DNA Breaks, Single-Stranded; DNA Primase; DNA Replication; DNA, Neoplasm; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Female; HEK293 Cells; Humans; Mice, Nude; Multifunctional Enzymes; Mutation; Neoplasms; Nucleic Acid Synthesis Inhibitors; Nucleotidyltransferases; Recombinational DNA Repair; Uracil-DNA Glycosidase; Xenograft Model Antitumor Assays; Mice
PubMed: 34508659
DOI: 10.1016/j.molcel.2021.08.016 -
Molecular Cell Oct 2021PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach...
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
Topics: Antineoplastic Agents; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; DNA Breaks, Single-Stranded; DNA Helicases; DNA Primase; DNA Repair; DNA Replication; DNA, Neoplasm; DNA-Binding Proteins; DNA-Directed DNA Polymerase; G2 Phase; Genomic Instability; HEK293 Cells; Humans; MRE11 Homologue Protein; Multifunctional Enzymes; Neoplasms; Nucleotidyltransferases; Proliferating Cell Nuclear Antigen; S Phase; Time Factors; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34624216
DOI: 10.1016/j.molcel.2021.09.013 -
Molecular Cell Aug 2023During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during...
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Replication; DNA Helicases; Saccharomyces cerevisiae; DNA, Single-Stranded
PubMed: 37506699
DOI: 10.1016/j.molcel.2023.06.035 -
Critical Reviews in Biochemistry and... Feb 2021DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication... (Review)
Review
DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The ase and DNA-directed ymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy.
Topics: Cell Nucleus; Cell Survival; Chromatin; DNA; DNA Damage; DNA Primase; DNA Replication; DNA-Directed DNA Polymerase; Gene Knockdown Techniques; Genomic Instability; Humans; Mitochondria; Multifunctional Enzymes
PubMed: 33179522
DOI: 10.1080/10409238.2020.1841089 -
Plants (Basel, Switzerland) Sep 2019Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some... (Review)
Review
Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some of the proteins that are required for these functions. The proteins and mechanisms for organelle DNA replication are very similar to bacterial or phage systems. The minimal replisome may consist of DNA polymerase, a primase/helicase, and a single-stranded DNA binding protein (SSB), similar to that found in bacteriophage T7. In , there are two genes for organellar DNA polymerases and multiple potential genes for SSB, but there is only one known primase/helicase protein to date. Genome copy number varies widely between type and age of plant tissues. Replication mechanisms are only poorly understood at present, and may involve multiple processes, including recombination-dependent replication (RDR) in plant mitochondria and perhaps also in chloroplasts. There are still important questions remaining as to how the genomes are maintained in new organelles, and how genome copy number is determined. This review summarizes our current understanding of these processes.
PubMed: 31546578
DOI: 10.3390/plants8100358