Genome-wide maps of UV damage repair and mutation suppression by CPD photolyase.

Ultraviolet (UV) light causes cyclobutane pyrimidine dimers (CPDs) and other DNA lesions that must be efficiently repaired to prevent cell death and mutagenesis. While mammals utilize the nucleotide excision repair (NER) pathway to repair CPDs, many species primarily utilize photolyase enzymes to repair UV damage. Our understanding of how different genomic and chromatin features impact photolyase repair across a eukaryotic genome is limited.

Defining APOBEC-induced mutation signatures and modifying activities in yeast.

APOBEC cytidine deaminases guard cells in a variety of organisms from invading viruses and foreign nucleic acids. Recently, several human APOBECs have been implicated in mutating evolving cancer genomes. Expression of APOBEC3A and APOBEC3B in yeast allowed experimental derivation of the substitution patterns they cause in dividing cells, which provided critical links to these enzymes in the etiology of the COSMIC single base substitution (SBS) signatures 2 and 13 in human tumors.

APOBEC3A deaminates CTG hairpin loops to promote fragility and instability of expanded CAG/CTG repeats.

CAG/CTG repeats are prone to expansion, causing several inherited human diseases. The initiating sources of DNA damage which lead to inaccurate repair of the repeat tract to cause expansions are not fully understood. Expansion-prone CAG/CTG repeats are actively transcribed and prone to forming stable R-loops with hairpin structures forming on the displaced single-stranded DNA (S-loops).

Contributing factors to the oxidation-induced mutational landscape in human cells.

8-oxoguanine (8-oxoG) is a common oxidative DNA lesion that causes G > T substitutions. Determinants of local and regional differences in 8-oxoG-induced mutability across genomes are currently unknown. Here, we show DNA oxidation induces G > T substitutions and insertion/deletion (INDEL) mutations in human cells and cancers.

Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U's) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U's can be directly replicated, resulting in C to T mutations, or U-DNA glycosylase can convert the U's to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs.

Network analysis of driver genes in human cancers.

Cancer is a heterogeneous disease that results from genetic alteration of cell cycle and proliferation controls. Identifying mutations that drive cancer, understanding cancer type specificities, and delineating how driver mutations interact with each other to establish disease is vital for identifying therapeutic vulnerabilities. Such cancer specific patterns and gene co-occurrences can be identified by studying tumor genome sequences, and networks have proven effective in uncovering relationships between sequences.

Hypomorphic mutation in the large subunit of replication protein A affects mutagenesis by human APOBEC cytidine deaminases in yeast.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils and function in antiviral innate immunity, RNA editing, and can cause hypermutation in chromosomes. The resulting uracils can be directly replicated, resulting in C to T mutations, or uracil-DNA glycosylase can convert the uracils to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depends on the origin of ssDNA mutagenized by APOBECs.

Her2 amplification, Rel-A, and Bach1 can influence APOBEC3A expression in breast cancer cells.

APOBEC-induced mutations occur in 50% of sequenced human tumors, with APOBEC3A (A3A) being a major contributor to mutagenesis in breast cancer cells. The mechanisms that cause A3A activation and mutagenesis in breast cancers are still unknown. Here, we describe factors that influence basal A3A mRNA transcript levels in breast cancer cells.

Genetic inhibitors of APOBEC3B-induced mutagenesis.

The cytidine deaminases APOBEC3A (A3A) and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we used a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast.

Contributions of replicative and translesion DNA polymerases to mutagenic bypass of canonical and atypical UV photoproducts.

UV exposure induces a mutation signature of C > T substitutions at dipyrimidines in skin cancers. We recently identified additional UV-induced AC > TT and A > T substitutions that could respectively cause BRAF V600K and V600E oncogenic mutations. The mutagenic bypass mechanism past these atypical lesions, however, is unknown.

Genetic modifiers of APOBEC-induced mutagenesis.

The cytidine deaminases APOBEC3A and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we utilized a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast.

Genome-wide maps of rare and atypical UV photoproducts reveal distinct patterns of damage formation and mutagenesis in yeast chromatin.

Ultraviolet (UV) light induces different classes of mutagenic photoproducts in DNA, namely cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and atypical thymine-adenine photoproducts (TA-PPs). CPD formation is modulated by nucleosomes and transcription factors (TFs), which has important ramifications for Ultraviolet (UV) mutagenesis. How chromatin affects the formation of 6-4PPs and TA-PPs is unclear.

The bile salt deoxycholate induces genetic point mutations that promote increased antibiotic resistance and fitness.

Oxidative damage to DNA is a significant source of mutations in living organisms. While DNA damage must be repaired to maintain the integrity of the genome and cell survival, errors made during DNA repair may contribute to evolution. Previous work has revealed that growth in the presence of bile salt deoxycholate (DOC) causes an increase in reactive oxygen species and the occurrence of 8-oxo-deoxyguanosine (8-oxo-dG) DNA lesions.

APOBEC-Induced Mutagenesis in Cancer.

The initiation, progression, and relapse of cancers often result from mutations occurring within somatic cells. Consequently, processes that elevate mutation rates accelerate carcinogenesis and hinder the development of long-lasting therapeutics. Recent sequencing of human cancer genomes has identified patterns of mutations, termed mutation signatures, many of which correspond to specific environmentally induced and endogenous mutation processes.

Characterization of sequence contexts that favor alternative end joining at Cas9-induced double-strand breaks.

Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end.

hMSH5 Regulates NHEJ and Averts Excessive Nucleotide Alterations at Repair Joints.

Inappropriate repair of DNA double-strand breaks (DSBs) leads to genomic instability, cell death, or malignant transformation. Cells minimize these detrimental effects by selectively activating suitable DSB repair pathways in accordance with their underlying cellular context. Here, we report that hMSH5 down-regulates NHEJ and restricts the extent of DSB end processing before rejoining, thereby reducing "excessive" deletions and insertions at repair joints.

Versatile Encapsulation and Synthesis of Potent Liposomes by Thermal Equilibration.

The wide-scale use of liposomal delivery systems is challenged by difficulties in obtaining potent liposomal suspensions. Passive and active loading strategies have been proposed to formulate drug encapsulated liposomes but are limited by low efficiencies (passive) or high drug specificities (active). Here, we present an efficient and universal loading strategy for synthesizing therapeutic liposomes.

dCas9 binding inhibits the initiation of base excision repair in vitro.

Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear.

The Shu complex prevents mutagenesis and cytotoxicity of single-strand specific alkylation lesions.

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes.

Single-stranded DNA binding proteins influence APOBEC3A substrate preference.

The cytidine deaminase, APOBEC3A (A3A), is a prominent source of mutations in multiple cancer types. These APOBEC-signature mutations are non-uniformly distributed across cancer genomes, associating with single-stranded (ss) DNA formed during DNA replication and hairpin-forming sequences. The biochemical and cellular factors that influence these specificities are unclear.

CTCF binding modulates UV damage formation to promote mutation hot spots in melanoma.

Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots.

Atypical UV Photoproducts Induce Non-canonical Mutation Classes Associated with Driver Mutations in Melanoma.

Somatic mutations in skin cancers and other ultraviolet (UV)-exposed cells are typified by C>T and CC>TT substitutions at dipyrimidine sequences; however, many oncogenic "driver" mutations in melanoma do not fit this UV signature. Here, we use genome sequencing to characterize mutations in yeast repeatedly irradiated with UV light. Analysis of ~50,000 UV-induced mutations reveals abundant non-canonical mutations, including T>C, T>A, and AC>TT substitutions.

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair.

Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells.