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1.
Genes (Basel) ; 14(1)2022 12 29.
Article in English | MEDLINE | ID: mdl-36672839

ABSTRACT

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.


Subject(s)
Flap Endonucleases , R-Loop Structures , Humans , DNA/genetics , DNA/metabolism , DNA Repair/genetics , Exonucleases/genetics , Flap Endonucleases/genetics , Flap Endonucleases/metabolism , Genomic Instability , RNA/genetics
2.
J Biol Chem ; 295(40): 13902-13913, 2020 10 02.
Article in English | MEDLINE | ID: mdl-32763971

ABSTRACT

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase ß (pol ß) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol ß loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol ß than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.


Subject(s)
DNA Polymerase beta/chemistry , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , Flap Endonucleases/chemistry , R-Loop Structures , Trinucleotide Repeats , Humans
3.
J Org Chem ; 84(6): 3624-3631, 2019 03 15.
Article in English | MEDLINE | ID: mdl-30806513

ABSTRACT

The Cu(I)- or Ag(I)-catalyzed cycloaddition between 8-ethynyladenine or guanine nucleosides and TMSN3 gave 8-(1- H-1,2,3-triazol-4-yl) nucleosides in good yields. On the other hand, reactions of 5-ethynyluracil or cytosine nucleosides with TMSN3 led to the chemoselective formation of triazoles via Cu(I)-catalyzed cycloaddition or vinyl azides via Ag(I)-catalyzed hydroazidation. These nucleosides with a minimalistic triazolyl modification showed excellent fluorescent properties with 8-(1- H-1,2,3-triazol-4-yl)-2'-deoxyadenosine (8-TrzdA), exhibiting a quantum yield of 44%. The 8-TrzdA 5'-triphosphate was incorporated into duplex DNA containing a one-nucleotide gap by DNA polymerase ß.


Subject(s)
Fluorescence , Purine Nucleosides/chemistry , Pyrimidine Nucleosides/chemistry , Triazoles/chemistry , Catalysis , Copper/chemistry , Molecular Structure , Purine Nucleosides/chemical synthesis , Pyrimidine Nucleosides/chemical synthesis , Silver/chemistry
4.
ACS Omega ; 3(4): 4276-4288, 2018 Apr 30.
Article in English | MEDLINE | ID: mdl-29732453

ABSTRACT

Transition-metal-catalyzed chlorosulfonylation of 5-ethynylpyrimidine nucleosides provided (E)-5-(ß-chlorovinyl)sulfones A, which undergo nucleophilic substitution with amines or thiols affording B. The treatment of vinyl sulfones A with ammonia followed by acid-catalyzed hydrolysis of the intermediary ß-sulfonylvinylamines gave 5-(ß-keto)sulfones C. The latter reacts with electrophiles, yielding α-carbon-alkylated or -sulfanylated analogues D. The 5'-triphosphates of A and C were incorporated into double-stranded DNA, using open and one-nucleotide gap substrates, by human or Escherichia coli DNA-polymerase-catalyzed reactions.

5.
PLoS One ; 12(5): e0177299, 2017.
Article in English | MEDLINE | ID: mdl-28475635

ABSTRACT

Trinucleotide repeat (TNR) instability is associated with human neurodegenerative diseases and cancer. Recent studies have pointed out that DNA base excision repair (BER) mediated by DNA polymerase ß (pol ß) plays a crucial role in governing somatic TNR instability in a damage-location dependent manner. It has been shown that the activities and function of BER enzymes and cofactors can be modulated by their polymorphic variations. This could alter the function of BER in regulating TNR instability. However, the roles of BER polymorphism in modulating TNR instability remain to be elucidated. A previous study has shown that a pol ß polymorphic variant, polßR137Q is associated with cancer due to its impaired polymerase activity and its deficiency in interacting with a BER cofactor, proliferating cell nuclear antigen (PCNA). In this study, we have studied the effect of the pol ßR137Q variant on TNR instability. We showed that pol ßR137Q exhibited weak DNA synthesis activity to cause TNR deletion during BER. We demonstrated that similar to wild-type pol ß, the weak DNA synthesis activity of pol ßR137Q allowed it to skip over a small loop formed on the template strand, thereby facilitating TNR deletion during BER. Our results further suggest that carriers with pol ßR137Q polymorphic variant may not exhibit an elevated risk of developing human diseases that are associated with TNR instability.


Subject(s)
DNA Polymerase beta/genetics , DNA Repair , Trinucleotide Repeats , DNA Damage , DNA Polymerase beta/metabolism , DNA Replication , Humans
6.
DNA Repair (Amst) ; 33: 24-34, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26123757

ABSTRACT

5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase ß (pol ß) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol ß wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol ß knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol ß plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol ß inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol ß, indicating a mutagenic effect induced by pol ß lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol ß generated an intermediate that failed to be extended by pol ß, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol ß plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol ß bypassing of a cdA lesion.


Subject(s)
DNA Breaks, Single-Stranded , DNA Polymerase beta/metabolism , DNA Repair , DNA Replication , Nucleosides/metabolism , Nucleotides/metabolism , Purines/metabolism , Animals , DNA/biosynthesis , DNA/metabolism , Flap Endonucleases/metabolism , Mice , Models, Biological , Nucleosides/chemistry , Purines/chemistry
7.
Nucleic Acids Res ; 43(12): 5948-60, 2015 Jul 13.
Article in English | MEDLINE | ID: mdl-25990721

ABSTRACT

Base excision repair (BER) of an oxidized base within a trinucleotide repeat (TNR) tract can lead to TNR expansions that are associated with over 40 human neurodegenerative diseases. This occurs as a result of DNA secondary structures such as hairpins formed during repair. We have previously shown that BER in a TNR hairpin loop can lead to removal of the hairpin, attenuating or preventing TNR expansions. Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3'-5' exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop. Coordinating with flap endonuclease 1, the APE1 3'-5' exonuclease activity cleaves the annealed upstream 3'-flap of a double-flap intermediate resulting from 5'-incision of an abasic site in the hairpin loop. Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.


Subject(s)
DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Trinucleotide Repeat Expansion , DNA/chemistry , DNA/metabolism , DNA Ligase ATP , DNA Ligases/metabolism , Exodeoxyribonucleases/metabolism , Flap Endonucleases/metabolism , Nucleic Acid Conformation
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