Modification-dependent restriction endonucleases are widely present in bacterial genomes and are thought to protect hosts from invading bacteriophages containing modified DNA (1
). Among many examples are the T-even phages, in which only 5-hydroxymethylcytosines (5hmC) are incorporated into the genome during replication and further modified to 5-glucosylhydroxymethylcytosine (5ghmC) by glucosyltransferases (1
). Although T4 wild-type DNA is resistant to most regular restriction enzymes, there are types of modification-dependent restriction enzymes that are able to restrict their infection in vivo
, including PvuRts1I (2
) among a few others. For a long time, the detailed in vitro
biochemical properties of PvuRts1I remained obscure (4
In mammalian genomes, it is commonly believed that 5-methylcytosine (5mC) is the major form of epigenetic base modification. Recently, the observation of 5hmC as the enzymatic oxidative product of 5mC in mammalian genomes (5
) has added an extra layer of complexity to the current understanding of epigenetic regulation and spurred rising interest in determining its genomic locations and metabolism. However, although the modified base 5hmC was discovered in bacteriophages >50 years ago (7
), there are few useful methods, either enzymatic or chemical, to specifically recognize 5hmc residues and pinpoint their locations in DNA, largely due to their close structural similarity to 5mC. For example, 5mC-dependent endonucleases, such as the the MspJI family (8
) or McrBC (9
), do not distinguish 5mC and 5hmC; 5mC-sensitive endonucleases, such as MspI or HpaII, etc.
, in most cases are equally affected by 5mC and 5hmC (4
). The widely used bisulfite conversion method cannot differentiate between 5mC and 5hmC and reports both forms indistinguishably (10
). Recently, the availability of 5hmC-specific antibodies has enabled a few enrichment-based methods [e.g. hMeDIP (12
)]. However, the format of the experiment, based on affinity pull-down, may limit the range of its application, and the resolution of the data is still far from base resolution.
Given mounting evidence for the importance of 5hmC in mammalian epigenetics and the previous experimental observations that PvuRts1I is able to specifically recognize 5hmC both in vivo
and in vitro
), we have set out to investigate the in vitro
biochemical properties of PvuRts1I and its homologs identified in REBASE (4
). During the course of our study, Szwagierczak et al.
) reported that recombinant PvuRts1I selectively cleaves 5hmC-containing DNA substrates and that the double-stranded cleavage sites are at N11–12
on the 3′-side of the recognized 5hmC site. In addition, the authors notice that PvuRts1I prefers to cleave at symmetric sites 5′-hm
C-5′, suggesting a likely in-cis
dimerization cleavage process (13
). Still, there are a number of questions left unanswered. For example, it is not clear whether PvuRts1I is applicable for mapping genomic 5hmC sites along with needing details concerning its practical use. In this regard, a quantitative description of substrate selectivity on 5hmC versus 5mC or unmodified cytosine is crucial, because in most human tissue DNA, the level of 5hmC is usually on the order of 0.01% of the total nucleotide (14
). During our investigation, we have observed that PvuRts1I is sensitive to different purification procedures, such that certain ions used in the buffer may quickly inactivate most of the enzyme in crude lysates (see ‘Results’ section). We have thus optimized purification conditions to obtain highly active enzymes. Furthermore, we have observed that in certain reaction conditions (e.g. reaction buffer or high enzyme concentration), PvuRts1I starts to digest 5mC and 5hmC indiscriminately ( in Results). This raises the concern of a possibly elevated false discovery rate if it is used improperly, which must be carefully monitored during its application.
Figure 3. Relative selectivity of PvuRts1I, PpeHI and AbaSDFI on unmodified cytosine (C), 5mC, 5hmC and 5ghmC (see ‘Materials and Methods’ section for description of methods). In each gel, the amount of enzyme is titrated from left (high) to right (more ...)
In this article, we systematically characterized the enzymatic properties of several members in the PvuRts1I family. In particular, we focus on comparing their substrate selectivity on different forms of cytosine modifications and evaluating their suitability in mapping genomic 5hmC sites. As one of the conclusions, we show that by using AbaSDFI, a homolog of PvuRts1I with much higher substrate selectivity, it is possible to map genomic 5hmC sites close to base resolution.