DNA Polymerase β:  Multiple Conformational Changes in the Mechanism of Catalysis†

Abstract DNA polymerase β (pol β) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol β lead to mutations, some of which occur within specific sequence contexts to generate mutation hotspots. The adenomatous polyposis coli (APC) gene is mutated within specific sequence contexts in colorectal carcinomas but the underlying mechanism is not fully understood. In previous work, we demonstrated that a somatic colon cancer variant of pol β, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol β within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol β catalysis is phosphodiester bond formation and suggest that substrate selection is governed at this step. Remarkably, we show that, unlike WT, a pre-catalytic step in the K289M pol β kinetic pathway becomes slower than phosphodiester bond formation with the APC DNA sequence but not with a different DNA substrate. Based on our studies, we propose that pre-catalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.

Download Full-text

DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

10.1101/306175 ◽

2018 ◽

Author(s):

Carel Fijen ◽

Mariam Mahmoud ◽

Rebecca Kaup ◽

Jamie Towle-Weicksel ◽

Joann Sweasy ◽

...

Keyword(s):

Dna Repair ◽

Dna Polymerase ◽

Single Molecule ◽

Conformational Changes ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Dna Polymerase Β ◽

Gapped Dna ◽

Dna Substrate

The eukaryotic DNA polymerase β plays an important role in cellular DNA repair as it fills gaps in single nucleotide gapped DNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially substrate binding and a fidelity-related conformational change called fingers closing. Here, we applied single-molecule Förster resonance energy transfer to study the conformational dynamics of Pol β. Using an acceptor labelled polymerase and a donor labelled DNA substrate, we measured distance changes associated with DNA binding and fingers movement. Our findings suggest that Pol β does not bend its gapped DNA substrate to the extent related crystal structures indicate: instead, bending seems to be significantly less profound. Furthermore, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. Additionally, we provide evidence that the fingers close only partially when an incorrect nucleotide is bound. This ajar conformation found in Pol β, a polymerase of the X-family, suggests the existence of an additional fidelity checkpoint similar to what has been previously proposed for a member of the A-family, the bacterial DNA polymerase I.

Download Full-text

Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011569 ◽

2019 ◽

Vol 295 (6) ◽

pp. 1613-1622

Author(s):

Mallory R. Smith ◽

Khadijeh S. Alnajjar ◽

Nicole M. Hoitsma ◽

Joann B. Sweasy ◽

Bret D. Freudenthal

Keyword(s):

Dna Polymerase ◽

Conformational Changes ◽

Dna Polymerases ◽

Catalytic Efficiency ◽

Dna Polymerase Β ◽

Replication Errors ◽

Human Dna ◽

Oxidatively Modified ◽

Dna Replication Errors

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2′-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Download Full-text

Faculty Opinions recommendation of Study of the lyase activity of human DNA polymerase β using analogues of the intermediate schiff base complex.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732995199.793545513 ◽

2018 ◽

Author(s):

Zucai Suo ◽

Andrew Reed

Keyword(s):

Schiff Base ◽

Dna Polymerase ◽

Schiff Base Complex ◽

Dna Polymerase Β ◽

Base Complex ◽

Human Dna ◽

Lyase Activity

Download Full-text

The human checkpoint sensor and alternative DNA clamp Rad9–Rad1–Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery

Biochemical Journal ◽

10.1042/bj20050211 ◽

2005 ◽

Vol 389 (1) ◽

pp. 13-17 ◽

Cited By ~ 41

Author(s):

Ekaterina SMIRNOVA ◽

Magali TOUEILLE ◽

Enni MARKKANEN ◽

Ulrich HÜBSCHER

Keyword(s):

Quality Control ◽

Dna Polymerase ◽

Base Excision Repair ◽

Excision Repair ◽

Dna Ligase ◽

Dna Polymerase Β ◽

Flap Endonuclease 1 ◽

Important Target ◽

Dna Ligase I ◽

Base Excision

The human checkpoint sensor and alternative clamp Rad9–Rad1–Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9–Rad1–Hus1 with MutY DNA glycosylase, DNA polymerase β and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9–Rad1–Hus1 complex, thus enhancing the quality control of DNA.

Download Full-text

DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities

DNA Repair ◽

10.1016/j.dnarep.2021.103050 ◽

2021 ◽

Vol 99 ◽

pp. 103050

Author(s):

Beverly A. Baptiste ◽

Stephanie L. Baringer ◽

Tomasz Kulikowicz ◽

Joshua A. Sommers ◽

Deborah L. Croteau ◽

...

Keyword(s):

Dna Polymerase ◽

Base Excision Repair ◽

Excision Repair ◽

Dna Polymerase Β ◽

Base Excision ◽

Dna Polymerase Γ

Download Full-text

Molecular disruption of DNA polymerase β for platinum sensitisation and synthetic lethality in epithelial ovarian cancers

Oncogene ◽

10.1038/s41388-021-01710-y ◽

2021 ◽

Author(s):

Reem Ali ◽

Adel Alblihy ◽

Islam M. Miligy ◽

Muslim L. Alabdullah ◽

Mansour Alsaleem ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Dna Polymerase ◽

Synthetic Lethality ◽

Ovarian Cancer Cells ◽

Dna Polymerase Β ◽

Mesenchymal Transition ◽

Platinum Sensitivity ◽

Cycle Arrest ◽

Ovarian Cancers

AbstractTargeting PARP1 [Poly(ADP-Ribose) Polymerase 1] for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase β (Polβ), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polβ deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polβ depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polβ small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG [Poly(ADP-Ribose) Glycohydrolase] inhibitor (PDD00017273) [but not PARP1 inhibitor (Olaparib)] was synthetically lethal in polβ deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD+) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polβ-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polβ targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.

Download Full-text