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Advances in Experimental Medicine and... 2017This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA... (Review)
Review
This chapter focuses on the enzymes and mechanisms involved in lagging-strand DNA replication in eukaryotic cells. Recent structural and biochemical progress with DNA polymerase α-primase (Pol α) provides insights how each of the millions of Okazaki fragments in a mammalian cell is primed by the primase subunit and further extended by its polymerase subunit. Rapid kinetic studies of Okazaki fragment elongation by Pol δ illuminate events when the polymerase encounters the double-stranded RNA-DNA block of the preceding Okazaki fragment. This block acts as a progressive molecular break that provides both time and opportunity for the flap endonuclease 1 (FEN1) to access the nascent flap and cut it. The iterative action of Pol δ and FEN1 is coordinated by the replication clamp PCNA and produces a regulated degradation of the RNA primer, thereby preventing the formation of long-strand displacement flaps. Occasional long flaps are further processed by backup nucleases including Dna2.
Topics: Animals; DNA; DNA Polymerase I; DNA Primase; DNA Primers; DNA Replication; Eukaryota; Eukaryotic Cells; Humans; Kinetics; RNA
PubMed: 29357056
DOI: 10.1007/978-981-10-6955-0_6 -
Biochemistry. Biokhimiia Aug 2020Many chemotherapy drugs block tumor cell division by damaging DNA. DNA polymerases eta (Pol η), iota (Pol ι), kappa (Pol κ), REV1 of the Y-family and zeta (Pol ζ) of... (Review)
Review
Many chemotherapy drugs block tumor cell division by damaging DNA. DNA polymerases eta (Pol η), iota (Pol ι), kappa (Pol κ), REV1 of the Y-family and zeta (Pol ζ) of the B-family efficiently incorporate nucleotides opposite a number of DNA lesions during translesion DNA synthesis. Primase-polymerase PrimPol and the Pol α-primase complex reinitiate DNA synthesis downstream of the damaged sites using their DNA primase activity. These enzymes can decrease the efficacy of chemotherapy drugs, contribute to the survival of tumor cells and to the progression of malignant diseases. DNA polymerases are promising targets for increasing the effectiveness of chemotherapy, and mutations and polymorphisms in some DNA polymerases can serve as additional prognostic markers in a number of oncological disorders.
Topics: Animals; Antineoplastic Agents; DNA; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Drug Resistance, Neoplasm; Humans; Neoplasms; Nucleic Acid Synthesis Inhibitors; Polymorphism, Genetic; Protein Biosynthesis
PubMed: 33045948
DOI: 10.1134/S0006297920080039 -
BioRxiv : the Preprint Server For... Oct 2023Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric...
Telomere maintenance requires extension of the G-rich telomeric repeat strand by telomerase and fill-in synthesis of the C-rich strand by Polα/Primase. Telomeric Polα/Primase is bound to Ctc1-Stn1-Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/Primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Phosphorylation of POT1 is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/Primase in an inactive auto-inhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/Primase into an active state that completes telomere replication through fill-in synthesis.
PubMed: 37215005
DOI: 10.1101/2023.05.08.539880 -
Nature Communications Aug 2023Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood....
Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRAS expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRAS-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRAS-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.
Topics: Humans; Ataxia Telangiectasia Mutated Proteins; Chromatin; DNA Primase; DNA-Directed DNA Polymerase; Genomic Instability; Heterochromatin; Multifunctional Enzymes; Proto-Oncogene Proteins p21(ras)
PubMed: 37591859
DOI: 10.1038/s41467-023-40578-2 -
Protein Science : a Publication of the... Jul 2017Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and... (Review)
Review
Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue.
Topics: DNA; DNA Helicases; DNA Replication; Nucleic Acid Heteroduplexes; RNA Helicases; RNA, Double-Stranded
PubMed: 28474797
DOI: 10.1002/pro.3187 -
The Enzymes 2016DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite... (Review)
Review
DNA replication in Escherichia coli initiates at oriC, the origin of replication and proceeds bidirectionally, resulting in two replication forks that travel in opposite directions from the origin. Here, we focus on events at the replication fork. The replication machinery (or replisome), first assembled on both forks at oriC, contains the DnaB helicase for strand separation, and the DNA polymerase III holoenzyme (Pol III HE) for DNA synthesis. DnaB interacts transiently with the DnaG primase for RNA priming on both strands. The Pol III HE is made up of three subassemblies: (i) the αɛθ core polymerase complex that is present in two (or three) copies to simultaneously copy both DNA strands, (ii) the β2 sliding clamp that interacts with the core polymerase to ensure its processivity, and (iii) the seven-subunit clamp loader complex that loads β2 onto primer-template junctions and interacts with the α polymerase subunit of the core and the DnaB helicase to organize the two (or three) core polymerases. Here, we review the structures of the enzymatic components of replisomes, and the protein-protein and protein-DNA interactions that ensure they remain intact while undergoing substantial dynamic changes as they function to copy both the leading and lagging strands simultaneously during coordinated replication.
Topics: DNA Polymerase III; DNA Primase; DNA Replication; DNA, Bacterial; Escherichia coli
PubMed: 27241927
DOI: 10.1016/bs.enz.2016.04.001 -
International Journal of Molecular... Nov 2020Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and...
Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.
Topics: DNA; DNA Primase; DNA-Directed DNA Polymerase; Flap Endonucleases; Humans; Manganese; Multifunctional Enzymes; Nuclear Proteins; RNA; Replication Protein A; Substrate Specificity
PubMed: 33261049
DOI: 10.3390/ijms21239027 -
Nature Cell Biology Jan 2022The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit...
The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Polα-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Polα) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Polα-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Polα-primase is the major effector of shieldin-dependent DSB processing.
Topics: Animals; BRCA1 Protein; Binding Sites; CRISPR-Cas Systems; Cell Line, Tumor; DNA; DNA Breaks, Double-Stranded; DNA Polymerase I; DNA Primase; DNA Repair; Gene Knockout Techniques; Humans; Mice; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Rad51 Recombinase; Shelterin Complex; Telomere-Binding Proteins; Tumor Suppressor p53-Binding Protein 1
PubMed: 35027730
DOI: 10.1038/s41556-021-00812-9 -
Cell May 2024Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At...
Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.
PubMed: 38838667
DOI: 10.1016/j.cell.2024.05.002 -
Nature May 2024Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two...
Prokaryotes have evolved intricate innate immune systems against phage infection. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB. GajA functions as a DNA endonuclease that is inactive in the presence of ATP. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Apoproteins; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; DNA; DNA Cleavage; Magnesium; Models, Molecular; Protein Binding; Protein Domains; Immunity, Innate; Microbial Viability; Bacillus cereus; Protein Structure, Quaternary; DNA Primase; DNA Topoisomerases
PubMed: 38471529
DOI: 10.1038/s41586-024-07270-x