The first human homologue of the E. coli MutY gene was cloned by Slupska et al. 37. By screening a human cDNA sequence database they identified an expressed sequence tag (EST) which was then used to probe a human bacterial artificial chromosome (BAC) library to isolate genomic human mutY, which was referred to as MUTYH, also called MYH. A full-length MUTYH cDNA clone from a human brain tissue cDNA library was also isolated which localized to the short arm of chromosome 1 between p32.1 and p34.3. MUTYH encompasses 7.1 kb and has 16 exons encoding a 535 amino acid protein displaying 41% identity with E. coli protein 37. Ohtsubo and co-workers 38 identified 10 forms of MUTYH transcripts, each with a different 5' sequence or first exon and each transcript being alternatively spliced, that were sub-grouped into 3 types, isoform MUTYHα (splice variants α1, 2, 3, and 4), isoform MUTYHβ (splice variants β1, 3, and 5), and isoform MUTYHγ (splice variants γ2, 3, and 4). The authors also showed that MUTYH protein encoded by type α mRNA possesses a mitochondrial targeting sequence (MTS), consisting of the amino terminal 14 residues which are required for its localization in the mitochondria 57, while those encoded by type β and γ mRNAs lack the MTS, and are localized in the nuclei. MUTYHα3 is the major isoform expressed in most cells and corresponds to the cDNA sequence isolated and characterized by Slupska et al. 37. In addition to human, homologues of MutY have been cloned from other mammals, including cow 58, mouse 59 and rat 60.

The open reading frame (ORF) of the full-length major isoform of MUTYH (MUTYHα3) encodes a 535 amino acid protein 38. This protein displays very high identity with MutY homologues from other mammals, 78 and 74.1% identity with mouse and rat MutY, respectively 61. Structurally, MUTYH reveals extensive homology and conservation with established structural domains found in E. coli MutY and many prokaryotic and eukaryotic BER enzymes, in addition to domains unique to MUTYH (Fig. 1).

MutY and its homologues contain the catalytic domain which shares several motifs with other glycosylases, including the helix-hairpin-helix (HhH), pseudo-HhH and an [4Fe-4S] iron sulphur cluster in the N terminus 6263. The latter has a high overall similarity to endonuclease III that excises oxidized pyrimidines 6465. HhH (aa 114–273 in MUTYH) functions to detect, recognize and remove adenines opposite to 8-oxoG by binding the phosphate backbone of the substrate. It includes a highly conserved aspartic acid residue (Asp222 in MUTYH), which is required for nucleophilic attack of the adenine base (reviewed in Ref. 61). MutY has a special carboxy-terminal domain that is not found in other BER glycosylases, with sequence and structural homology to MutT (an 8-oxoGTPase), and this domain has an important role in the recognition of 8-oxoG because the truncation of the domain results in loss of discrimination between 8-oxoG:A and G:A mispairs 666768. In addition, ORF of MUTYH also contains other domains that are shown to be the binding sites of other proteins. By using HeLa nuclear extracts, Parker and co-workers 69 demonstrated that MUTYH contains an APE1 binding site (aa 300), a replication protein A (RPA) binding site (aa 6–32), and a PCNA binding site (aa 505–527).

The primary function of MUTYH as a BER DNA glycosylase is to excise adenines or 2-hydroxy-adenines (2-OH-A) misincorporated opposite 7,8-dihydro-8-oxo-guanine (8-oxoG or GO) 70. GO is one of the most stable products of DNA damage resulting from reactive oxygen species (ROS) 71 because this oxidized form of the guanine base can pair with adenine as well as cytosine with equal frequency during DNA replication and thus has the potential to cause a high rate of G:C to T:A transversions 7273. In E. coli MutY, together with MutM and MutT (formamidopyrimidine-DNA glycosylase: FPG), play important roles in reducing the mutagenic effects of GO lesions 7475. MutM removes GO paired with cytosine and introduces a single strand gap as a result of the accompanying AP-lyase activity 75; whereas MutT hydrolyzes 8-oxo-dGTP and depletes it from the nucleotide pool 73. Similar DNA error avoiding mechanisms have also been shown in human cells, whereby MTH1 (MutT homologue), OGG1 (MutM homologue), and MUTYH (MutY homologue) have been proposed to function in the reduction of GO in human genome 76.

Yang and co-workers demonstrated that the cloned cDNA of MUTYH 3777 complement the mutator phenotype of a MutY E. coli strain and prevented G:C to T:A transversions 78. Suppressive activities of MUTYH against G:C to T:A transversions have also been shown in human cells in vivo 79.

Unlike MTH1 or OGG1, MUTYH possesses no detectable AP-lyase activity 78. The authors also demonstrated that APE1 catalytic activity is required for the formation of cleaved AP DNA and stimulation of MUTYH glycosylase activity by increasing the formation of the MUTYH-DNA complex. Similar findings were also reported by Parker and co-workers, who showed that MUTYH interacts with APE1, PCNA, and RPA, suggesting a role in long-patch BER 69. Despite the structural homology between MUTYH and its bacterial counterpart, the human MUTYH protein efficiently removes 2-OH-A from 2-OH-A:G mismatches 38, while the bacterial protein removes 2-OH-A from the substrate containing 2-OH-A:G pairs very poorly 80 (Fig. 2, 3).

Recent findings from experiments employing full-length structure of MutY cross-linked to DNA containing 8-oxoG have shed light on the mechanism of recognition and removal of 8-oxoG:A mismatched by MutY. Although the precise mechanism is still unclear, the results of these experiments suggest that MutY relies heavily on the recognition of GO to locate A bases for excision. For example, Fromme and co-workers 68 demonstrated that in the X-ray crystal structure of an inactive variant (Asp144Asn) of B. stearothermophilus MutY there are extensive contacts with 8-oxoG but minimal contacts with adenine. In another instance Bernards and co-workers 82 conducted time-resolved fluorescence experiments of the MutY A-excision reaction using 8-oxo·GA substrates and found a multiphase reaction profile, with a fast process being associated with changes at 8-oxoG and a slower process associated with altering the environment of the adenine. MutY exposes its target by deeply penetrating the DNA helix, interrupting helical stacking on both strands, encircling the DNA with its catalytic core and MutT-like domains, and rotating the phosphodiester bonds surrounding the nucleotide, causing the target base adenine to be flipped out of the DNA helix 6883. The iron-sulphur cluster [4Fe-4S] contained in the catalytic core of MutY plays a significant role in facilitating DNA binding with the damaged region and catalyzing the removal of adenine 84. This iron-sulphur cluster plays a DNA-dependent electron transfer role which enables MutY to quickly and efficiently seek out damaged sites in the genome using DNA-mediated charge transport 85.

Evidence is accumulating in the literature implicating the interactions of DNA glycosylases and non-BER pathways, although in most cases the details of the mechanism are not well understood 86. In addition to their participation in BER, DNA glycosylases have been reported to interact with nucleotide excision repair (NER) and mismatch repair (MMR).

MUTYH-initiated BER and MMR pathways share common features in terms of function and timing of action. Functionally, both pathways participate in repairing DNA lesions resulting from DNA oxidation. Both pathways take place immediately after DNA replication to increase the fidelity of DNA replication and bear a task to distinguish newly synthesized DNA strands from their parental counterparts 3787888990919293.

The molecular evidence of the interaction of MUTYH and MMR proteins was first obtained from an elegant study by Gu and co-workers 94. In their experiment they found that hMUTYH directly interacts with hMSH6 in the hMSH2/hMSH6 (hMutSα) heterodimer, which functions to bind to the mismatches and initiate the repair on the daughter DNA strands 95. They also observed that this physical protein interaction can occur in the absence of DNA. Based on their findings, the authors further hypothesized that proteins involved in DNA replication, mismatch repair and base excision repair may exist as a multiple-protein complex and that hMUTYH may be orientated in the replication fork to recognize 8-oxoG on the parental strands and to excise misincorporated A on the daughter strand 94. This hypothesis is supported by some evidence. Both hMUTYH and hMSH6 interact with replication proteins PCNA and RPA 699697. Moreover, both proteins show overexpression during S phase and colocalize with PCNA at replication foci 698796.

Although to date there is no direct evidence for the physical interactions between MUTYH and proteins involved in NER, there is evidence indicating that DNA glycosylases are coupled to the NER pathway, more specifically to the subpathway transcription-coupled repair (TCR).

One example is the repair of thymine glycols which are normally repaired by thymine glycol DNA glycosylase. Leadon and Cooper 98 reported that thymine glycols generated in NER- and BER-proficient human cells following ionizing radiation exposure are removed in a biased fashion from the transcribed strand of an expressed gene. In this case, TCR of thymine glycols exists in the same locus in cells where BER is active, suggesting that the thymine glycol-DNA glycosylase is coupled to TCR. It is logical therefore that the question arises whether such coupled repair exists for other lesions that are normally repaired by BER glycosylases.

It has been proposed that the crucial event in transcription-coupled NER (TC-NER) is the stalling of an elongating RNA polymerase II upon a lesion, which recruits the repair proteins to the damage site 99100101102. Therefore, the ability of a lesion on the transcribed strand to block the RNA polymerase transcription complex has been assumed to be crucial for TCR. Kathe and co-workers 103 found that DNA base damage does not block transcriptional elongation by RNA polymerase II in HeLa cell nuclear extracts, but single-strand breaks do. It is known that single-strand breaks are common BER processing intermediates. It is therefore tempting to speculate that a strand break produced by a DNA glycosylase at an oxidative lesion in a transcription bubble would serve as a block to the RNA polymerase apparatus which in turn will signal the TC-NER to take place.

Cheadle and Sampson 104 have presented a comprehensive review of MUTYH variants and their diagnostic implications. They reported that as of the end of 2006, 30 mutations that are predicted to produce truncated proteins have been reported in MUTYH, consisting of 11 nonsense, 9 small insertion/deletions and 10 splice site variants. Moreover, 52 missense variants and three small inframe insertion/deletions have been reported that are distributed throughout the gene 104. To date, as presented in Table 2, the list of reported MUTYH variants has grown. Of the reported variants, Y165C and G382D together account for approximately 73% of reported MUTYH variants 104 and have been commonly identified in Caucasian populations, including American, British, Danish, Finnish, Dutch, Italian, and Portuguese (reviewed in refs. 61105). Beside these two variants, the occurrence of the rest of the variants is rare, although recurrent variants have been observed in some populations and will be discussed later in this section.