Trinucleotide repeat instability via DNA base excision repair.

Trinucleotide repeat (TNR) instability is the cause of over 40 human neurodegenerative diseases and certain types of cancer. TNR instability can result from DNA replication, repair, recombination, and gene transcription. Emerging evidence indicates that DNA base damage and base excision repair (BER) play an active role in regulating somatic TNR instability. These processes may potentially modulate the onset and progression of TNR-related diseases, given that TNRs are hotspots of DNA base damage that are present in mammalian cells with a high frequency. In this review, we discuss the recent advances in our understanding of the molecular mechanisms underlying BER-mediated TNR instability. We initially discuss the roles of the BER pathway and locations of DNA base lesions in TNRs and their interplay with non-B form DNA structures in governing repeat instability. We then discuss how the coordinated activities of BER enzymes can modulate a balance between the removal and addition of TNRs to regulate somatic TNR instability. We further discuss how this balance can be disrupted by the crosstalk between BER and DNA mismatch repair (MMR) machinery resulting in TNR expansion. Finally, we suggest future directions regarding BER-mediated somatic TNR instability and its association with TNR disease prevention and treatment.

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase ß and a de novo DNA Methyltransferase.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from -189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase ß (pol ß). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol ß and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.

The NIH Common Fund/Roadmap Epigenomics Program: Successes of a comprehensive consortium.

The NIH Roadmap Epigenomics Program was launched to deliver reference epigenomic data from human tissues and cells, develop tools and methods for analyzing the epigenome, discover novel epigenetic marks, develop methods to manipulate the epigenome, and determine epigenetic contributions to diverse human diseases. Here, we comment on the outcomes from this program: the scientific contributions made possible by a consortium approach and the challenges, benefits, and lessons learned from this group science effort.

An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract.

Oxidative DNA damage and base excision repair (BER) play important roles in modulating trinucleotide repeat (TNR) instability that is associated with human neurodegenerative diseases and cancer. We have reported that BER of base lesions can lead to TNR instability. However, it is unknown if modifications of the sugar in an abasic lesion modulate TNR instability. In this study, we characterized the effects of the oxidized sugar, 5'-(2-phosphoryl-1,4-dioxobutane)(DOB) in CAG repeat tracts on the activities of key BER enzymes, as well as on repeat instability. We found that DOB crosslinked with DNA polymerase ß and inhibited its synthesis activity in CAG repeat tracts. Surprisingly, we found that DOB also formed crosslinks with DNA ligase I and inhibited its ligation activity, thereby reducing the efficiency of BER. This subsequently resulted in the accumulation of DNA strand breaks in a CAG repeat tract. Our study provides important new insights into the adverse effects of an oxidized abasic lesion on BER and suggests a potential alternate repair pathway through which an oxidized abasic lesion may modulate TNR instability.

Dynamics of p53: A Master Decider of Cell Fate.

Cellular stress-induced temporal alterations-i.e., dynamics-are typically exemplified  by the dynamics of p53 that serve as a master to determine cell fate. p53 dynamics were initially  identified as the variations of p53 protein levels. However, a growing number of studies have  shown that p53 dynamics are also manifested in variations in the activity, spatial location, and  posttranslational modifications of p53 proteins, as well as the interplay among all p53 dynamical  features. These are essential in determining a specific outcome of cell fate. In this review, we  discuss the importance of the multifaceted features of p53 dynamics and their roles in the cell fate  decision process, as well as their potential applications in p53-based cancer therapy. The review  provides new insights into p53 signaling pathways and their potentials in the development of new  strategies in p53-based cancer therapy.

Proliferating cell nuclear antigen prevents trinucleotide repeat expansions by promoting repeat deletion and hairpin removal.

DNA base lesions and base excision repair (BER) within trinucleotide repeat (TNR) tracts modulate repeat instability through the coordination among the key BER enzymes DNA polymerase ß, flap endonuclease 1 (FEN1) and DNA ligase I (LIG I). However, it remains unknown whether BER cofactors can also alter TNR stability. In this study, we discovered that proliferating cell nuclear antigen (PCNA), a cofactor of BER, promoted CAG repeat deletion and removal of a CAG repeat hairpin during BER in a duplex CAG repeat tract and CAG hairpin loop, respectively. We showed that PCNA stimulated LIG I activity on a nick across a small template loop during BER in a duplex (CAG)20 repeat tract promoting small repeat deletions. Surprisingly, we found that during BER in a hairpin loop, PCNA promoted reannealing of the upstream flap of a double-flap intermediate, thereby facilitating the formation of a downstream flap and stimulating FEN1 cleavage activity and hairpin removal. Our results indicate that PCNA plays a critical role in preventing CAG repeat expansions by modulating the structures of dynamic DNA via cooperation with BER enzymes. We provide the first evidence that PCNA prevents CAG repeat expansions during BER by promoting CAG repeat deletion and removal of a TNR hairpin.

Crosstalk between MSH2-MSH3 and polß promotes trinucleotide repeat expansion during base excision repair.

Studies in knockout mice provide evidence that MSH2-MSH3 and the BER machinery promote trinucleotide repeat (TNR) expansion, yet how these two different repair pathways cause the mutation is unknown. Here we report the first molecular crosstalk mechanism, in which MSH2-MSH3 is used as a component of the BER machinery to cause expansion. On its own, pol ß fails to copy TNRs during DNA synthesis, and bypasses them on the template strand to cause deletion. Remarkably, MSH2-MSH3 not only stimulates pol ß to copy through the repeats but also enhances formation of the flap precursor for expansion. Our results provide direct evidence that MMR and BER, operating together, form a novel hybrid pathway that changes the outcome of TNR instability from deletion to expansion during the removal of oxidized bases. We propose that cells implement crosstalk strategies and share machinery when a canonical pathway is ineffective in removing a difficult lesion.

AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair.

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.

Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol ß DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol ß then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.