DNA Base Excision Repair Intermediates Influence Duplex–Quadruplex Equilibrium

Sowers, Mark L.; Conrad, James W.; Chang-Gu, Bruce; Cherryhomes, Ellie; Hackfeld, Linda; Sowers, Lawrence C.

doi:10.3390/molecules28030970

Cited by 4 publications

(3 citation statements)

References 49 publications

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“…Therefore, if OGG1 does bind and subsequently resolve the G4 before cleaving the N-glycosyl bond, APE1 likely re-folds the G4 structure while bound. Moreover, prior work has shown that APE1 activity is greatly inhibited by G4 DNA despite tight binding affinities for the structure Sowers et al, 2023). Because pre-steady-state kinetics enable the resolution of distinct key catalytic steps (i.e., DNA cleavage and product release) and we utilized double-stranded AP-G4 VEGF promoter substrates, the work here expands on these prior observations.…”

Section: Ligation Depends On Fen1mentioning

confidence: 78%

TheVEGFG-quadruplex forming promoter is repaired via long-patch BER

Hussen,

Kravitz,

Freudenthal

et al. 2023

Preprint

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In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased ~2-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

show abstract

Section: Ligation Depends On Fen1mentioning

confidence: 78%

TheVEGFG-quadruplex forming promoter is repaired via long-patch BER

Hussen,

Kravitz,

Freudenthal

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Therefore, if OGG1 does bind and subsequently resolve the G4 before cleaving the N‐glycosyl bond, APE1 likely re‐folds the G4 structure while bound. Moreover, prior work has shown that APE1 activity is greatly inhibited by G4 DNA despite tight binding affinities for the structure (Fleming et al, 2021; Sowers et al, 2023). Because pre‐steady‐state kinetics enable the resolution of distinct key catalytic steps (i.e., DNA cleavage and product release) and we utilized double‐stranded AP‐G4 VEGF promoter substrates, the work here expands on these prior observations.…”

Section: Discussionmentioning

confidence: 99%

Oxidative DNA damage on the VEGF G‐quadruplex forming promoter is repaired via long‐patch BER

Hussen,

Kravitz,

Freudenthal

et al. 2023

Environ and Mol Mutagen

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In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B‐form and G4 DNA conformations, resulting in epigenetic‐like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre‐steady‐state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double‐stranded VEGF promoter G4 DNA substrates. Moreover, pre‐steady‐state kinetics revealed that compared to B‐form DNA, APE1 repair activity on the G4 was decreased ~two‐fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substrates via strand displacement DNA synthesis in contrast to a single insertion on B‐form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short‐patch BER. Instead, repair requires the long‐patch BER flap‐endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

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“…However, the C250 and C228 mutation hotspots in the hTERT promoter are not at CpG sites, and therefore 5 mC deamination and repair would not explain why these sites are mutational hotspots. While uracil repair within the hTERT promoter has not been investigated, it is known that the completion of DNA repair by the base excision-repair pathway can be inhibited when uracil bases are located within DNA sequences like telomeres that can form non-duplex structures [ 19 , 20 , 21 ]. Inefficient BER within the hTERT promoter could contribute to increased mutation frequencies.…”

Section: Introductionmentioning

confidence: 99%

Transition Mutations in the hTERT Promoter Are Unrelated to Potential i-motif Formation in the C-Rich Strand

Conrad,

Sowers,

Yap

et al. 2023

Biomolecules

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Increased expression of the human telomere reverse transcriptase (hTERT) in tumors promotes tumor cell survival and diminishes the survival of patients. Cytosine-to-thymine (C-to-T) transition mutations (C250T or C228T) in the hTERT promoter create binding sites for transcription factors, which enhance transcription. The G-rich strand of the hTERT promoter can form G-quadruplex structures, whereas the C-rich strand can form an i-motif in which multiple cytosine residues are protonated. We considered the possibility that i-motif formation might promote cytosine deamination to uracil and C-to-T mutations. We computationally probed the accessibility of cytosine residues in an i-motif to attack by water. We experimentally examined regions of the C-rich strand to form i-motifs using pH-dependent UV and CD spectra. We then incubated the C-rich strand with and without the G-rich complementary strand DNA under various conditions, followed by deep sequencing. Surprisingly, deamination rates did not vary substantially across the 46 cytosines examined, and the two mutation hotspots were not deamination hotspots. The appearance of mutational hotspots in tumors is more likely the result of the selection of sequences with increased promoter binding affinity and hTERT expression.

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DNA Base Excision Repair Intermediates Influence Duplex–Quadruplex Equilibrium

Cited by 4 publications

References 49 publications

TheVEGFG-quadruplex forming promoter is repaired via long-patch BER

TheVEGFG-quadruplex forming promoter is repaired via long-patch BER

Oxidative DNA damage on the VEGF G‐quadruplex forming promoter is repaired via long‐patch BER

Transition Mutations in the hTERT Promoter Are Unrelated to Potential i-motif Formation in the C-Rich Strand

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