Naked DNA Substrates Research Articles

The lyase activity of bifunctional DNA glycosylases and the 3'-diesterase activity of APE1 contribute to the repair of oxidized bases in nucleosomes.

The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be ‘handed’ from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3′ diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.

Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge

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.

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

The activity of uracil DNA glycosylases (UDGs), which recognize and excise uracil bases from DNA, has been well characterized on naked DNA substrates but less is known about activity in chromatin. We therefore prepared a set of model nucleosome substrates in which single thymidine residues were replaced with uracil at specific locations and a second set of nucleosomes in which uracils were randomly substituted for all thymidines. We found that UDG efficiently removes uracil from internal locations in the nucleosome where the DNA backbone is oriented away from the surface of the histone octamer, without significant disruption of histone-DNA interactions. However, uracils at sites oriented toward the histone octamer surface were excised at much slower rates, consistent with a mechanism requiring spontaneous DNA unwrapping from the nucleosome. In contrast to the nucleosome core, UDG activity on DNA outside the core DNA region was similar to that of naked DNA. Association of linker histone reduced activity of UDG at selected sites near where the globular domain of H1 is proposed to bind to the nucleosome as well as within the extra-core DNA. Our results indicate that some sites within the nucleosome core and the extra-core (linker) DNA regions represent hot spots for repair that could influence critical biological processes.

Evidence That Nucleosomes Inhibit Mismatch Repair in Eukaryotic Cells

The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSalpha and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSalpha specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSalpha displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSalpha sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.

ATP-Dependent Chromatin Remodeling Is Required for Base Excision Repair in Conventional but Not in Variant H2A.Bbd Nucleosomes

In eukaryotes, base excision repair (BER) is responsible for the repair of oxidatively generated lesions. The mechanism of BER on naked DNA substrates has been studied in detail, but how it operates on chromatin remains unclear. Here we have studied the mechanism of BER by introducing a single 8-oxo-7,8-dihydroguanine (8-oxoG) lesion in the DNA of reconstituted positioned conventional and histone variant H2A.Bbd nucleosomes. We found that 8-oxoguanine DNA glycosylase, apurinic/apyrimidinic endonuclease, and polymerase beta activities were strongly reduced in both types of nucleosomes. In conventional nucleosomes SWI/SNF stimulated the processing of 8-oxoG by each one of the three BER repair factors to efficiencies similar to those for naked DNA. Interestingly, SWI/SNF-induced remodeling, but not mobilization of conventional nucleosomes, was required to achieve this effect. A very weak effect of SWI/SNF on the 8-oxoG BER removal in H2A.Bbd histone variant nucleosomes was observed. The possible implications of our data for the understanding of in vivo mechanisms of BER are discussed.

Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

The nonhomologous end joining pathway for DNA double strand break repair requires Ku to bind DNA ends and subsequently recruit other nonhomologous end joining factors, including the DNA-dependent protein kinase catalytic subunit and the XRCC4-Ligase IV complex, to the break site. Ku loads at a break by threading the DNA ends through a circular channel in its structure. This binding mechanism explains both the high specificity of Ku for ends and its ability to translocate along DNA once loaded. However, DNA in cells is typically coated with other proteins (e.g. histones), which might be expected to block the ability of Ku to load in this manner. Here we address how the nature of a protein obstruction dictates how Ku interacts with a DNA end. Ku is unable to access the ends within an important intermediate in V(D)J recombination (a complex of RAG proteins bound to cleaved recombination targeting signals), but Ku readily displaces the linker histone, H1, from DNA. Ku also retains physiological affinity for nucleosome-associated ends. Loading onto nucleosome-associated ends still occurs by threading the end through its channel, but rather than displacing the nucleosome, Ku peels as much as 50 bp of DNA away from the histone octamer surface. We suggest a model where Ku utilizes an unusual characteristic of its three-dimensional structure to recognize certain protein-occluded ends without the extensive remodeling of chromatin structure required by other DNA repair pathways.

Human ACF1 Alters the Remodeling Strategy of SNF2h

The human ACF chromatin-remodeling complex (hACF) contains the ATPase motor protein SNF2h and the non-catalytic hACF1 subunit. Here, we have compared the ability of SNF2h and a reconstituted hACF complex containing both SNF2h and hACF1 to remodel a series of nucleosomes containing different lengths of DNA overhang. Both SNF2h and hACF functioned in a manner consistent with sliding a canonical nucleosome. However, the non-catalytic subunit, hACF1, altered the remodeling properties of SNF2h by changing the nature of the requirement for a DNA overhang in the nucleosomal substrate and altering the DNA accessibility profile of the remodeled products. Surprisingly, addition of hACF1 to SNF2h increased the amount of DNA overhang needed to observe measurable amounts of DNA accessibility, but decreased the amount of overhang needed for a measurable binding interaction. We propose that these hACF1 functions might contribute to making the hACF complex more efficient at nucleosome spacing compared with SNF2h. In contrast, the SWI/SNF complex and its ATPase subunit BRG1 generated DNA accessibility profiles that were similar to each other, but different significantly from those of hACF and SNF2h. Thus, we observed divergent remodeling behaviors in these two remodeling families and found that the manner in which hACF1 alters the remodeling behavior of the ATPase is not shared by SWI/SNF subunits.

Increased Bending Rigidity of Single DNA Molecules by H-NS, a Temperature and Osmolarity Sensor

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed λ-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15–20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32°C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

Lashings of DNA methylation, forkfuls of chromatin remodeling.

Epigenetic control of gene expression in animals and plants is often correlated with changes in cytosine methylation at specific chromosomal loci. This leads to the irreversible promoter silencing of many genes, transposons, and endogenous retroviruses (Bestor 2000; Martienssen and Colot 2001). How DNA methylation patterns are set up during development, especially in the context of chromatin, is not well understood. Cytosine methyltransferases catalyze the transfer of a methyl group onto the C-5 position of cytosine from the universal cofactor SAM (Bestor 2000). Two main types of methyltransferase activity exist in mammals: a de novo activity and a maintenance activity. Dnmt3a and Dnmt3b have been identified as de novo methyltransferases, which methylate cytosine at CpG dinucleotides on both strands with little sequence specificity (Okano et al. 1998; Lyko et al. 1999). Dnmt1 is predominantly a postreplicative maintenance methyltransferase, which recognizes hemimethylated substrates, and acts to restore methylated cytosines at CpGs on the newly duplicated strand (Lyko et al. 1999; Bestor 2000). Mutation studies in mice have shown that the methyltransferase genes are necessary for embryonic (Dnmt1 and Dnmt3b) and postnatal (Dnmt3a) development (Lei et al. 1996; Okano et al. 1999). All three enzymes have been shown to methylate naked DNA substrates in vitro. However, complicating the issue is the in vivo evidence suggesting that patterns of DNA methylation are established and maintained within a nucleosomal infrastructure (Bird and Wolffe 1999). As yet we know little about the mechanistic basis of Dnmt3a and Dnmt3b activity in vivo, but in the case of Dnmt1 it has been shown that this maintenance methyltransferase enzyme can be targeted to PCNA, an auxiliary component of the DNA replication complex (Chuang et al. 1997). It is probable that chromatin assembly precedes DNA methylation behind the replication fork. Assembly of DNA into nucleosomes can prevent access by methyltransferases to their in vivo substrates (Kladde and Simpson 1994). Yet in the genome, methylated CpGs are distributed with equal probability either in the DNA wrapped around the nucleosome or in the linker region (Reik et al. 2001).

Cleavage Preferences of the Apoptotic Endonuclease DFF40 (Caspase-activated DNase or Nuclease) on Naked DNA and Chromatin Substrates

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.

Strand Exchange Through a DNA-Protein Complex Requires a DNA Helicase

The phage T4 uvsX and gene 32 proteins are capable of mediating homologous strand exchange, a central reaction in general genetic recombination, in vitro using naked DNA substrates. However, strand exchange is blocked by a sequence specific DNA-protein complex. Since protein-complexed substrates must be employed in vivo, this suggests that another factor(s) is required for strand exchange with protein-complexed DNAs. We show here that a DNA helicase, the T4 dda protein, allows the phage recombination machinery to drive branch migration through a RNA polymerase-promoter complex. This is the first observation of in vitro strand exchange using protein-bound substrates. These results suggest that a DNA helicase is a necessary component of the "protein machine" that mediates recombination in vivo.

DNA repair defect in xeroderma pigmentosum group C and complementing factor from HeLa cells.

A predominant form of the inherited syndrome xeroderma pigmentosum is genetic complementation group C (XP-C). XP-C cells are defective in DNA nucleotide excision repair in the bulk of the genome but can repair transcribed strands of active genes. An activity that can complement the repair deficiency of extracts from XP-C cells has been purified approximately 2,000-fold from HeLa cells. The factor also increases the unscheduled DNA synthesis of XP-C fibroblasts in vivo after microinjection. Hydrodynamic measurements show that the XP-C complementing factor has a native molecular mass of approximately 160 kDa. The factor binds tightly to single-stranded DNA cellulose, eluting in approximately 1.3 M NaCl. No incision or ATPase activity of the protein alone was detected. XP-C protein is involved in an early stage of repair since its presence was required before the start of gap-filling repair synthesis. In vitro complementation was achieved with naked DNA substrates, and so a primary role in processing chromatin to allow access for repair enzymes seems unlikely. Surprisingly, however, extracts from an XP-C cell line introduced some incisions in UV-irradiated DNA; these were unstable in cell extracts and did not lead to complete repair. The data can be explained by a model in which XP-C factor participates in forming one of the repair incisions flanking DNA damage but not the other. In transcribed DNA, its role is subsumed by RNA polymerase and/or transcription coupling factors.