The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome, MYH-associated polyposis (MAP). A key feature of MYH activity is its coordination with the cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector between the catalytic and 8-oxoG recognition-domains of hMYH (IDC, residues 295-350) is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH (SpMyh1) as a model system. In vitro studies of SpMyh1 identified residues I 261 and E 262 of the IDC (equivalent to V 315 and E 316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.

Mtr4p belongs to the Ski2p family of DEVH-box containing proteins and is required for processing and degradation of a variety of RNA substrates in the nucleus. In particular, Mtr4p is required for creating the 5.8 S ribosomal RNA from its 7 S precursor, proper 3-end processing of the U4 small nuclear RNA and some small nucleolar RNAs, and degradation of aberrant mRNAs and tRNAs. In these studies we have shown that Mtr4p has RNA-dependent ATPase (or dATPase) activity that is stimulated effectively by likely substrates (e.g. tRNA) but surprisingly weakly by poly(A). Using an RNA strand-displacement assay, we have demonstrated that Mtr4p can, in the presence of ATP or dATP, unwind the duplex region of a partial duplex RNA substrate in the 335direction. We have examined the ability of Mtr4p to bind model RNA substrates in the presence of nucleotides that mimic the stages (i.e. ATP-bound, ADP-bound, and nucleotide-free) of the unwinding reaction. Our results demonstrate that the presence of a non-hydrolyzable ATP analog allows Mtr4p to discriminate between partial duplex RNA substrates, binding a 3-tailed substrate with 5-fold higher affinity than a 5-tailed substrate. In addition, Mtr4p displays a marked preference for binding to poly(A) RNA relative to an oligoribonucleotide of the same length and a random sequence. This binding exhibits apparent cooperativity and different dynamic behavior from binding to the random single-stranded RNA. This unique binding mode might be employed primarily for degradation.The processing of newly transcribed RNAs is the initial post-transcriptional series of events that culminates in an expressed gene product. Ribosomal, small nuclear, small nucleolar, and messenger RNAs are transcribed as long precursors (pre-RNAs) that must be fragmented and trimmed to yield functional RNAs (1). In particular, each ribosomal RNA, small nuclear RNA, and small nucleolar RNA must have its 3Ј-end exonucleolytically shortened to remove any deleterious or otherwise unnecessary extensions. Any byproducts of the conversion from extended transcripts to functional RNAs must be rapidly degraded, and defective RNAs must be efficiently removed from circulation by 3Ј35Ј exonucleolytic degradation. From an extensive analysis of nuclear RNA processing in Saccharomyces cerevisiae (2-4), it has become clear that this 3Ј-end maturation is mediated by the DExH-box helicase Mtr4p and the nuclear exosome, a collection of six RNase PH homologues (Rrp41p, Rrp42p, Rrp43p, Rrp45p, Rrp46p, and Mtr3p) that are apparently inactive (5), three proteins (Rrp4p, Rrp40p, and Csl4p) that form a cap on the RNase PH hexamer (6), and two active 3Ј35Ј exonucleases (Rrp44p (5, 7) and Rrp6p (8)). Mtr4p is an indispensable partner of the exosome that presumably maintains the momentum of exonucleolytic decay/processing as it moves through structured regions of its substrates.

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G°) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.

Ordered assembly of Ag receptor genes by VDJ recombination is a key determinant of successful lymphocyte differentiation and function. Control of gene rearrangement has been traditionally viewed as a result of complex reorganization of the nucleochromatin mediated by several nuclear factors. Selective recombination of the variable (V) genes to the diversity (D), but not joining (J), gene segments within the TCRβ locus has been shown to be controlled by recombination signal (RS) sequences that flank the gene segments. Through ex vivo and in vitro recombination assays, we demonstrate that the Rag proteins can discriminate between the RS of the D and J genes and enforce selective D gene incorporation into the TCRβ variable domain in the absence of other nuclear factors or chromatin structure. DNA binding studies indicate that discrimination is not simply caused by higher affinity binding of the Rag proteins to the isolated 12RS of the D as opposed to the J genes. Furthermore, we also demonstrate that the 12RS within the TCRβ locus is functionally inferior to the consensus 12RS. We propose that selective gene segment usage is controlled at the level of differential assembly and/or stability of synaptic RS complexes, and that evolutionary “deterioration” of the RS motifs may have been important to allow the VDJ recombinase to exert autonomous control over gene segment use during gene rearrangement.