Structural basis for recruitment of replicative DNA polymerase to PCNA

DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.

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Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

Nucleic Acids Research ◽

10.1093/nar/gkab799 ◽

2021 ◽

Author(s):

Keisuke Oki ◽

Mariko Nagata ◽

Takeshi Yamagami ◽

Tomoyuki Numata ◽

Sonoko Ishino ◽

...

Keyword(s):

Crystal Structure ◽

Dna Polymerase ◽

Direct Interaction ◽

Crystal Structure Analysis ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Functional Connection ◽

Terminal Domain ◽

The Family ◽

Lagging Strand

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

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Structure of the processive human Pol δ holoenzyme

10.1101/872879 ◽

2019 ◽

Author(s):

Claudia Lancey ◽

Muhammad Tehseen ◽

Vlad-Stefan Raducanu ◽

Fahad Rashid ◽

Nekane Merino ◽

...

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Structural Basis ◽

Catalytic Core ◽

Regulatory Subunits ◽

Functional Interactions ◽

Proliferating Cell ◽

Lagging Strand ◽

Catalytic Cleft

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ’s activity in replicating the human genome.

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Structure of telomerase-bound CST with Polymerase α-Primase

10.1101/2021.12.28.474374 ◽

2021 ◽

Author(s):

Yao He ◽

Song He ◽

Henry Chan ◽

Yaqiang Wang ◽

Baocheng Liu ◽

...

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Structural Basis ◽

Binding Motif ◽

Telomerase Reverse Transcriptase ◽

Telomeric Dna ◽

Double Stranded Dna ◽

Key Players ◽

Single Complex ◽

Linear Chromosomes

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3'-overhang of the G-strand and a group of proteins called shelterin. Among these, TPP1 and POT1 associate with the 3'-overhang, with POT1 binding the G-strand and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The ends of the telomeric DNA are replicated and maintained by telomerase, for the G-strand, and subsequently DNA Polymerase α-Primase (PolαPrim), for the C-strand. PolαPrim is stimulated by CTC1-STN1-TEN1 (CST), but the structural basis of both PolαPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolαPrim, as well as of PolαPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolαPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex-telomerase core RNP, p50/TPP1, CST and PolαPrim-that provides unprecedented insights into CST and PolαPrim recruitment and handoff between G-strand and C-strand synthesis.

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Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion

Biochemical Journal ◽

10.1042/bcj20190906 ◽

2020 ◽

Vol 477 (5) ◽

pp. 937-951

Author(s):

Hala Ouzon-Shubeita ◽

Caroline K. Vilas ◽

Seongmin Lee

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Cisplatin Resistance ◽

Structural Basis ◽

Abasic Site ◽

Dna Polymerase Β ◽

Dna Lesion ◽

Human Dna ◽

Optimal Conformation ◽

Structural Insights

The cisplatin-1,2-d(GpG) (Pt-GG) intrastrand cross-link is the predominant DNA lesion generated by cisplatin. Cisplatin has been shown to predominantly induce G to T mutations and Pt-GG permits significant misincorporation of dATP by human DNA polymerase β (polβ). In agreement, polβ overexpression, which is frequently observed in cancer cells, is linked to cisplatin resistance and a mutator phenotype. However, the structural basis for the misincorporation of dATP opposite Pt-GG is unknown. Here, we report the first structures of a DNA polymerase inaccurately bypassing Pt-GG. We solved two structures of polβ misincorporating dATP opposite the 5′-dG of Pt-GG in the presence of Mg2+ or Mn2+. The Mg2+-bound structure exhibits a sub-optimal conformation for catalysis, while the Mn2+-bound structure is in a catalytically more favorable semi-closed conformation. In both structures, dATP does not form a coplanar base pairing with Pt-GG. In the polβ active site, the syn-dATP opposite Pt-GG appears to be stabilized by protein templating and pi stacking interactions, which resembles the polβ-mediated dATP incorporation opposite an abasic site. Overall, our results suggest that the templating Pt-GG in the polβ active site behaves like an abasic site, promoting the insertion of dATP in a non-instructional manner.

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