Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O6-alkylguanine DNA-alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the AGT reactive cysteine and alkyltransferase activity. Here we determine S. pombe ATL structures without and with damaged DNA containing endogenous lesion O6-methylguanine or cigarette smoke-derived O6-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to XPG and ERCC1 in S. pombe homologs Rad13 and Swi10 and biochemical interactions with UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

O 6 -alkylguanine-DNA alkyltransferase (AGT) is a ubiquitous enzyme with an amino acid sequence that is conserved in Eubacteria, Archaea and Eukarya. It repairs O 6 -alkylguanine and O 4 -alkylthymine adducts in single-stranded and duplex DNAs. In performing these functions, AGT must partition between adduct-containing sites and the large excess of adduct-free DNA distributed throughout the genome. Here we characterize the binding of human AGT to linear double-stranded, adduct-free DNAs ranging in length from 11 bp to 2686 bp. Moderately-cooperative binding (22.6 ± 3.7 ≤ ω ≤ 145.0 ± 37.0) results in an all-or-nothing association pattern on short templates. The apparent binding site size S app (mean = 4.39 ± 0.02 bp) oscillates with increasing template length. Oscillations in cooperativity factor ω have the same frequency but are of opposite phase to S app , with the result that the most stable protein-protein and protein-DNA interactions occur at the highest packing densities. The oscillation period (4.05 ± 0.02 bp/protein) is nearly identical to the occluded binding site size obtained at the highest measured binding density (4 bp/protein) and is significantly smaller than the contour length (~8 bp) occupied in crystalline complexes. A model in which protein molecules overlap along the DNA contour is proposed to account for these features. High AGT densities resulting from cooperative binding may allow efficient search for lesions in the context of chromatin remodeling and DNA replication. O 6 -alkylguanine and O 4 -alkylthymine are mutagenic and carcinogenic adducts that are formed in DNA as a result of exposure to alkylating agents [1][2][3]. Both adducts are repaired by the ubiquitous repair protein, O 6 -alkylguanine-DNA alkyltransferase (AGT) 1 [1,4,5]. Ironically, AGT also protects cells against chemotherapeutic drugs that methylate or chloroethylate DNA [6,7]. Clinical trials of AGT-inhibitors are underway in an attempt to increase the efficacy of such drugs in cancer chemotherapy [8][9][10]. In addition, overexpression of AGT in hematopoietic stem cells is under study as a means to protect normal cells against the † Supported by NIH grants GM-070662 (to M.G.F.) and CA-97209 (to A.E.P.) and Medical Scientist Training Program Grant 5 T32 GM-08601-05 (J.J.R.).

SUMMARY The mutagenic and cytotoxic effects of many alkylating agents are reduced by O6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multi-protein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a 3-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n + 3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein crosslinking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions.

Recent studies implicate Poly (ADP-ribose) polymerase 1 (PARP1) in alternative splicing regulation, and PARP1 may be an RNA-binding protein. However, detailed knowledge of RNA targets and the RNA-binding region for PARP1 are unknown. Here we report the first global study of PARP1–RNA interactions using PAR–CLIP in HeLa cells. We identified a largely overlapping set of 22 142 PARP1–RNA-binding peaks mapping to mRNAs, with 20 484 sites located in intronic regions. PARP1 preferentially bound RNA containing GC-rich sequences. Using a Bayesian model, we determined positional effects of PARP1 on regulated exon-skipping events: PARP1 binding upstream and downstream of the skipped exons generally promotes exon inclusion, whereas binding within the exon of interest and intronic regions closer to the skipped exon promotes exon skipping. Using truncation mutants, we show that removal of the Zn1Zn2 domain switches PARP1 from a DNA binder to an RNA binder. This study represents a first step into understanding the role of PARP1–RNA interaction. Continued identification and characterization of the functional interplay between PARPs and RNA may provide important insights into the role of PARPs in RNA regulation.