David S. Pederson

Rules of Engagement for Base Excision Repair in Chromatin

Most of the DNA in eukaryotes is packaged in tandemly arrayed nucleosomes that, together with numerous DNA‐ and nucleosome‐associated enzymes and regulatory factors, make up chromatin. Chromatin modifying and remodeling agents help regulate access to selected DNA segments in chromatin, thereby facilitating transcription and DNA replication and repair. Studies of nucleotide excision repair (NER), single strand break repair (SSBR), and the homology‐directed repair (HDR), and non‐homologous end‐joining (NHEJ) double strand break repair pathways have led to an “access‐repair‐restore” paradigm, in which chromatin in the vicinity of damaged DNA is disrupted, thereby enabling efficient repair and the subsequent repackaging of DNA into nucleosomes. When damage is extensive, these repair processes are accompanied by cell cycle checkpoint activation, which provides cells with sufficient time to either complete the repair or initiate apoptosis. It is not clear, however, if base excision repair (BER) of the ∼20,000 or more oxidative DNA damages that occur daily in each nucleated human cell can be viewed through this same lens. Until recently, we did not know if BER requires or is accompanied by nucleosome disruption, and it is not yet clear that anything short of overwhelming oxidative damage (resulting in the shunting of DNA substrates into other repair pathways) results in checkpoint activation. This review highlights studies of how oxidatively damaged DNA in nucleosomes is discovered and repaired, and offers a working model of events associated with BER in chromatin that we hope will have heuristic value. J. Cell. Physiol. 228: 258–266, 2013.

Nucleosome Disruption by DNA Ligase III-XRCC1 Promotes Efficient Base Excision Repair

ABSTRACT Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase β (Pol β), which catalyze the first three steps in BER, are able to process their substrates in both 601- and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase IIIα-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase IIIα-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol β on nucleosome substrates. Collectively, these findings provide insights into rate-limiting steps that govern BER in chromatin and reveal a unique role for ligase IIIα-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes.

Initiation of Base Excision Repair of Oxidative Lesions in Nucleosomes by the Human, Bifunctional DNA Glycosylase NTH1

ABSTRACT Oxidative lesions account for much of the spontaneously occurring DNA damage in normal cells and, left unrepaired, can be mutagenic or cytotoxic. We have investigated the capacity of purified human enzymes to initiate the base excision repair (BER) of oxidative lesions in model nucleosomes. In a construct where the minor groove of a thymine glycol lesion faced outward from the histone octamer, the human DNA glycosylase NTH1 (hNTH1) processed the lesion with nearly the same efficiency as in naked DNA. The hNTH1 reaction did not generate free DNA, indicating that the first step in BER occurred without irreversibly disrupting nucleosomes. Instead, lesion processing entailed the formation of nucleosome-hNTH1 ternary complexes that could be visualized in a gel mobility shift assay. These complexes contained both processed and unprocessed DNA. hNTH1 processing of lesions whose minor groove faced toward the histone octamer was poor at low hNTH1 concentrations but increased substantially as hNTH1 concentrations increased to nearly physiological levels. Additionally, an inward-facing lesion near the nucleosome edge was more efficiently processed than one closer to the nucleosome dyad. These observations suggest that access to sterically occluded lesions entails the partial, reversible unwrapping of DNA from the histone octamer, allowing hNTH1 to capture its DNA substrate when it is in an unwound state.

Non-specific DNA binding interferes with the efficient excision of oxidative lesions from chromatin by the human DNA glycosylase, NEIL1

Although DNA in eukaryotes is packaged in nucleosomes, it remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway, which begins with the excision of damaged bases by DNA glycosylases. We reported recently that the human DNA glycosylase hNTH1 (human Endonuclease III), a member of the HhH GpG superfamily of glycosylases, can excise thymine glycol lesions from nucleosomes without requiring or inducing nucleosome disruption; optimally oriented lesions are excised with an efficiency approaching that seen for naked DNA [1]. To determine if this property is shared by human DNA glycoylases in the Fpg/Nei family, we investigated the activity of NEIL1 on defined nucleosome substrates. We report here that the cellular concentrations and apparent k(cat)/K(M) ratios for hNTH1 and NEIL1 are similar. Additionally, after adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities toward nucleosome substrates. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. As a result, hNTH1 is able to excise both accessible and sterically occluded lesions from nucleosomes at physiological concentrations, while the high non-specific DNA affinity of NEIL1 would likely hinder its ability to process sterically occluded lesions in cells. These results suggest that, in vivo, NEIL1 functions either at nucleosome-free regions (such as those near replication forks) or with cofactors that limit its non-specific binding to DNA.

Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin

All organisms suffer double‐strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non‐homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individuals mutation burden and risk of cancer. This review describes radiological, chemical, and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome‐specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis). J. Cell. Physiol. 230: 3–14, 2016.

Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae.

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

Contribution of DNA unwrapping from histone octamers to the repair of oxidatively damaged DNA in nucleosomes.

Reactive oxygen species generate ~20,000 oxidative lesions in the DNA of every cell, every day. Most of these lesions are located within nucleosomes, which package DNA in chromatin and impede base excision repair (BER). We demonstrated previously that periodic, spontaneous partial unwrapping of DNA from the underlying histone octamer enables BER enzymes to bind to oxidative lesions that would otherwise be sterically inaccessible. In the present study, we asked if these periodic DNA unwrapping events are frequent enough to account for the estimated rates of BER in vivo. We measured rates of excision of oxidative lesions from sites in nucleosomes that are accessible only during unwrapping episodes. Using reaction conditions appropriate for presteady-state kinetic analyses, we derived lesion exposure rates for both 601 and 5S rDNA-based nucleosomes. Although DNA unwrapping-mediated exposure of a lesion ~16NT from the nucleosome edge occurred ~7-8 times per minute, exposure rates fell dramatically for lesions located 10 or more NT further in from the nucleosome edge. The rates likely are too low to account for observed rates of BER in cells. Thus, chromatin remodeling, either BER-specific or that associated with transcription, replication, or other DNA repair processes, probably contributes to efficient BER in vivo.

The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages

Background: Ionizing radiation can produce clustered lesions in DNA; attempted base excision repair of these lesions can generate double-strand breaks (DSBs). Results: The extent to which nucleosomes suppress DSB formation is governed by their structural and dynamic properties. Conclusion: Nucleosomes suppress formation of radiation-induced DSBs. Significance: This study helps elucidate mechanisms responsible for potentially mutagenic or lethal DSBs. Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.

Evidence That Partial Unwrapping of DNA from Nucleosomes Facilitates the Binding of Heat Shock Factor following DNA Replication in Yeast

In the yeast Saccharomyces cerevisiae, heat shock transcription factor (HSF) binds heat shock element (HSE) DNA shortly after DNA replication, independently of its activation by heat shock. To determine if HSF binding occurs before newly replicated DNA is packaged into nucleosomes, we inserted an HSE into a DNA segment that normally forms a positioned nucleosome in vivo. Transcription from constructs designed to create steric competition between binding of HSF and histone H2A-H2B dimers was generally poor, suggesting that nucleosome assembly precedes and inhibits HSF binding. However, one such construct was as transcriptionally active as a nucleosome-free control. Structural analyses suggested that ∼40 base pairs of DNA, including the HSE, had unwrapped from the 3′ edge of the histone octamer, allowing HSF to bind; ∼100 base pairs remained in association with the histone octamer, with the same translational and rotational orientation as was seen for the poorly transcribed constructs. Modeling studies suggest that the active and inactive constructs differ from one another in the ease with which the HSE and flanking sequences can adopt the curvature needed to form a stable nucleosome. These differences may influence the probability of DNA unwrapping from already assembled nucleosomes and the subsequent binding of HSF.