During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on ...nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.
•There are 4 major ATP-dependent chromatin remodeling (ACR) families in eukaryotes.•ACR is responsible for regulation of transcription, replication, and some repair processes.•BER is responsible for ...the repair of most chemical modifications to DNA bases.•ACR activity facilitates BER through protein expression and opening of chromatin.•There lacks evidence of directed ACR activity to accommodate BER access to DNA.
Base Excision Repair (BER) is a conserved, intracellular DNA repair system that recognizes and removes chemically modified bases to insure genomic integrity and prevent mutagenesis. Aberrant BER has been tightly linked with a broad spectrum of human pathologies, such as several types of cancer, neurological degeneration, developmental abnormalities, immune dysfunction and aging. In the cell, BER must recognize and remove DNA lesions from the tightly condensed, protein-coated chromatin. Because chromatin is necessarily refractory to DNA metabolic processes, like transcription and replication, the compaction of the genomic material is also inhibitory to the repair systems necessary for its upkeep. Multiple ATP-dependent chromatin remodelling (ACR) complexes play essential roles in modulating the protein-DNA interactions within chromatin, regulating transcription and promoting activities of some DNA repair systems, including double-strand break repair and nucleotide excision repair. However, it remains unclear how BER operates in the context of chromatin, and if the chromatin remodelling processes that govern transcription and replication also actively regulate the efficiency of BER. In this review we highlight the emerging role of ACR in regulation of BER.
DNA damage in chromatin comes in many forms, including single base lesions that induce base excision repair (BER). We and others have shown that the structural location of DNA lesions within ...nucleosomes greatly influences their accessibility to repair enzymes. Indeed, a difference in the location of uracil as small as one-half turn of the DNA backbone on the histone surface can result in a 10-fold difference in the time course of its removal in vitro. In addition, the cell has evolved several interdependent processes capable of enhancing the accessibility of excision repair enzymes to DNA lesions in nucleosomes, including post-translational modification of histones, ATP-dependent chromatin remodeling and interchange of histone variants in nucleosomes. In this review, we focus on different factors that affect accessibility of BER enzymes to nucleosomal DNA.
The Fanconi anemia (FA) molecular network consists of 15 "FANC" proteins, of which 13 are associated with mutations in patients with this cancer-prone chromosome instability disorder. Whereas ...historically the common phenotype associated with FA mutations is marked sensitivity to DNA interstrand crosslinking agents, the literature supports a more global role for FANC proteins in coping with diverse stresses encountered by replicative polymerases. We have attempted to reconcile and integrate numerous observations into a model in which FANC proteins coordinate the following physiological events during DNA crosslink repair: (a) activating a FANCM-ATR-dependent S-phase checkpoint, (b) mediating enzymatic replication-fork breakage and crosslink unhooking, (c) filling the resulting gap by translesion synthesis (TLS) by error-prone polymerase(s), and (d) restoring the resulting one-ended double-strand break by homologous recombination repair (HRR). The FANC core subcomplex (FANCA, B, C, E, F, G, L, FAAP100) promotes TLS for both crosslink and non-crosslink damage such as spontaneous oxidative base damage, UV-C photoproducts, and alkylated bases. TLS likely helps prevent stalled replication forks from breaking, thereby maintaining chromosome continuity. Diverse DNA damages and replication inhibitors result in monoubiquitination of the FANCD2-FANCI complex by the FANCL ubiquitin ligase activity of the core subcomplex upon its recruitment to chromatin by the FANCM-FAAP24 heterodimeric translocase. We speculate that this translocase activity acts as the primary damage sensor and helps remodel blocked replication forks to facilitate checkpoint activation and repair. Monoubiquitination of FANCD2-FANCI is needed for promoting HRR, in which the FANCD1/BRCA2 and FANCN/PALB2 proteins act at an early step. We conclude that the core subcomplex is required for both TLS and HRR occurring separately for non-crosslink damages and for both events during crosslink repair. The FANCJ/BRIP1/BACH1 helicase functions in association with BRCA1 and may remove structural barriers to replication, such as guanine quadruplex structures, and/or assist in crosslink unhooking.
Nucleosomes affect Cas9 binding and activity at on-target sites, but their impact at off-target sites is unknown. To investigate how nucleosomes affect Cas9 cleavage at off-target sites in vitro, we ...used a single guide RNA (sgRNA) that has been previously shown to efficiently direct Cas9 cleavage at the edge of the strongly positioned 601 nucleosome. Our data indicate that single mismatches between the sgRNA and DNA target have relatively little effect on Cas9 cleavage of naked DNA substrates, but strongly inhibit cleavage of nucleosome substrates, particularly when the mismatch is in the sgRNA “seed” region. These findings indicate that nucleosomes may enhance Cas9 specificity by inhibiting cleavage of off-target sites at the nucleosome edge.
Histones play a crucial role in the organization of DNA in the nucleus, but their presence can prevent interactions with DNA binding proteins responsible for repair of DNA damage. Uracil is an ...abundant mutagenic lesion recognized by uracil DNA glycosylase (UDG) in the first step of base excision repair (BER). In nucleosome core particles (NCPs), we find substantial differences in UDG-directed cleavage at uracils rotationally positioned toward (U-In) or away from (U-Out) the histone core, or midway between these orientations (U-Mid). Whereas U-Out NCPs show a cleavage rate just below that of naked DNA, U-In and U-Mid NCPs have markedly slower rates of cleavage. Crosslinking of U-In DNA to histones in NCPs yields a greater reduction in cleavage rate but, surprisingly, yields a higher rate of cleavage in U-Out NCPs compared with uncrosslinked NCPs. Moreover, the next enzyme in BER, APE1, stimulates the activity of human UDG in U-Out NCPs, suggesting these enzymes interact on the surface of histones in orientations accessible to UDG. These data indicate that the activity of UDG likely requires "trapping" transiently exposed states arising from the rotational dynamics of DNA on histones.
DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage ...occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method,
-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data show that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs ARS binding factor 1 (Abf1) and rDNA enhancer binding protein 1 (Reb1), but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of ultraviolet (UV) damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.
The versatility of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing makes it a popular tool for many research and biotechnology applications. Recent ...advancements in genome editing in eukaryotic organisms, like fungi, allow for precise manipulation of genetic information and fine-tuned control of gene expression. Here, we provide an overview of CRISPR genome editing technologies in yeast, with a particular focus on
. We describe the tools and methods that have been previously developed for genome editing in
and discuss tips and experimental tricks for promoting efficient, marker-free genome editing in this model organism. These include sgRNA design and expression, multiplexing genome editing, optimizing Cas9 expression, allele-specific editing in diploid cells, and understanding the impact of chromatin on genome editing. Finally, we summarize recent studies describing the potential pitfalls of using CRISPR genome targeting in yeast, including the induction of background mutations.
Homologous recombination repair (HRR) encompasses mechanisms that employ homologous DNA sequences as templates for repair or tolerance of a wide range of DNA lesions that inhibit DNA replication in S ...phase. Arguably the most imposing of these DNA lesions is that of the interstrand crosslink (ICL), consisting of a covalently attached chemical bridge between opposing DNA strands. ICL repair requires the coordinated activities of HRR and a number of proteins from other DNA repair and damage response systems, including nucleotide excision repair, base excision repair, mismatch repair, and translesion DNA synthesis (TLS). Interestingly, different organisms favor alternative methods of HRR in the ICL repair process. E. coli perform ICL repair using a homology-driven damage bypass mechanism analogous to daughter strand gap repair. Eukaryotes from yeast to humans initiate ICL repair primarily during DNA replication, relying on HRR activity to restart broken replication forks associated with double-strand break intermediates induced by nucleolytic activities of other excision repair factors. Higher eukaryotes also employ several additional factors, including members of the Fanconi anemia damage-response network, which further promote replication-associated ICL repair through the activation and coordination of various DNA excision repair, TLS, and HRR proteins. This review focuses on the proteins and general mechanisms of HRR associated with ICL repair in different model organisms. Environ. Mol. Mutagen., 2010.
PDF
