Crystal structure of thymine DNA glycosylase conjugated to SUMO-1

Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) plays essential roles in eukaryotic growth and development. Many covalently modified SUMO targets have been identified; however, the extent and significance of noncovalent interactions of SUMO with cellular proteins is poorly understood. Here, large-scale yeast two-hybrid screens repeatedly identified a surprisingly small number of proteins that interacted with three Arabidopsis SUMO isoforms. These SUMO-interacting proteins are nuclear and fall into two main categories: six histone or DNA methyltransferses or demethylases and six proteins that we show to be the evolutionary and functional homologs of SUMOtargeted ubiquitin ligases (STUbLs). The selectivity of the screen for several methylases and demethylases suggests that SUMO interaction with these proteins has a significant impact on chromatin methylation. Furthermore, the Arabidopsis STUbLs (AT-STUbLs) complemented to varying degrees the growth defects of the Schizosaccharomyces pombe STUbL mutant rfp1/rfp2, and three of them also complemented the genome integrity defects of this mutant, demonstrating that these proteins show STUbL activity. We show that one of the AT-STUbLs least related to the S. pombe protein, AT-STUbL4, has acquired a plant-specific function in the floral transition. It reduces protein levels of CYCLING DOF FACTOR 2, hence increasing transcript levels of CONSTANS and promoting flowering through the photoperiodic pathway.SIM | Slx5/Slx8 | Nucleolus | Flowering time | CDF

Section: Resultsmentioning

confidence: 72%

mentioning

confidence: 99%

Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions

Elrouby

Bonequi

Porri

et al. 2013

“…Such a destabilizing electrostatic interaction could occur if the His151 imidazole ring is neutral and N δ1 carries a lone electron pair (i.e., >N: rather than >N-H). Accordingly, the other His151 imidazole nitrogen (N ε2 ) provides a short hydrogen bond (2.8 Å) to the backbone oxygen of Pro125, an interaction observed in all three previous structures of TDG (29,43,44). Notably, a neutral rather than protonated His151 imidazole would likely be best suited for excision of fC and caC, which have an NH 2 rather than carbonyl oxygen at C4 of the nucleobase.…”

Section: Resultsmentioning

confidence: 99%

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

Maiti

Noon

MacKerell

et al. 2012

168

DNA base excision repair is essential for maintaining genomic integrity and for active DNA demethylation, a central element of epigenetic regulation. A key player is thymine DNA glycosylase (TDG), which excises thymine from mutagenic G·T mispairs that arise by deamination of 5-methylcytosine (mC). TDG also removes 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC produced by Tet enzymes. Recent studies show that the glycosylase activity of TDG is essential for active DNA demethylation and for embryonic development. Our understanding of how repair enzymes excise modified bases without acting on undamaged DNA remains incomplete, particularly for mismatch glycosylases such as TDG. We solved a crystal structure of TDG (catalytic domain) bound to a substrate analog and characterized active-site residues by mutagenesis, kinetics, and molecular dynamics simulations. The studies reveal how TDG binds and positions the nucleophile (water) and uncover a previously unrecognized catalytic residue (Thr197). Remarkably, mutation of two active-site residues (Ala145 and His151) causes a dramatic enhancement in G·T glycosylase activity but confers even greater increases in the aberrant removal of thymine from normal A·T base pairs. The strict conservation of these residues may reflect a mechanism used to strike a tolerable balance between the requirement for efficient repair of G·T lesions and the need to minimize aberrant action on undamaged DNA, which can be mutagenic and cytotoxic. Such a compromise in G·T activity can account in part for the relatively weak G·T activity of TDG, a trait that could potentially contribute to the hypermutability of CpG sites in cancer and genetic disease. D NA base excision repair (BER) plays an established role in maintaining genomic integrity, and recent studies indicate that BER is also essential for active DNA demethylation, a key element of epigenetic gene regulation (1-3). A central player in both processes is thymine DNA glycosylase (TDG), which initiates BER by excising damaged or modified forms of 5-methylcytosine (mC) that arise at 5′-CpG-3′ sites. TDG was discovered for its ability to selectively remove T from G·T mispairs, mutagenic lesions that can arise from deamination of mC to T (4, 5). TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidized forms of mC that are generated by Tet enzymes (6, 7). In addition, TDG was shown to be essential for active DNA demethylation and for embryonic development (8, 9), a role that likely involves excision of deaminated or oxidized forms of mC generated by a deaminase or Tet enzyme (7,(10)(11)(12). Thus, the ability of TDG to remove deaminated and oxidized forms of mC is important for genetic and epigenetic integrity.The question of how DNA glycosylases remove modified bases without acting upon the huge excess of undamaged DNA remains largely unresolved, particularly for mismatch glycosylases such as TDG and MBD4 (methyl binding domain IV) (13-15). These enzymes face the formidable task of removing a normal base, t...

“…SUMO-1 K39 and K46 show large chemical shifts in complexes with peptides derived from PIASx, PML, and SAE2 (21). Furthermore, the cocrystal structure of thymidine DNA glycosylase with SUMO-1 shows that SUMO-1 K39 and K37 make contact with a 307DVQEV311 motif in thymidine DNA glycosylase (22). Moreover, the cocrystal of SUMO-1 with RanGAP1, RanBP2͞NUP358, and UBC-9 reveals that RanBP2 D2631 is hydrogen bonded to SUMO-1 K39, with RanBP2-SUMO-1 contacts spanning a 2631DVLIV2635 motif in RanBP2 (23).…”

Section: Discussionmentioning

confidence: 99%

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

Rosendorff

Sakakibara

et al. 2006

104

100

Small ubiquitin-like modifier (SUMO) modification of transcription factors is generally associated with repression. Reverse genetic analysis of SUMO-1, and -2 conserved residues emphasized the importance of dual charge reversals in abrogating the critical role of SUMO-2 K33, K35, and K42 in repression. GST-SUMO-2-affinity chromatography followed by liquid chromatography (LC)-MS analysis identified proteins that appeared to bind preferentially to WT SUMO-2 versus SUMO-2 K33E and K35E. LSD1, NXP-2, KIAA0809 (ARIP4), SAE2, RanGAP1, PELP1, and SETDB1 bound to SUMO-2 and not to SUMO-2 K33E, K42E, or K35E and K42E. Although LSD1 is a histone lysine demethylase, and histone H3K4 was demethylated at a SUMO-2-repressed promoter, neither overexpression of a dominant-negative LSD1 nor LSD1 depletion with RNA interference affected SUMO-2-mediated repression, indicating that LSD1 is not essential for repression, in this context. When tethered to a promoter by fusion to Gal4, NXP-2 repressed transcription, consistent with a role for NXP-2 in SUMO-mediated repression. SUMO-2-associated proteins identified in this study may contribute to SUMO-dependent regulation of transcription or other processes.regulation ͉ repressor ͉ ubiquitin-like S mall ubiquitin-like modifier (SUMO) is a ubiquitin-like protein that is reversibly covalently attached to lysines in target proteins, resulting in altered protein function, localization, or stability. SUMO modification of corepressors, coactivators, histones, histone-modifying enzymes, or sequence-specific transcription factors usually down-regulates transcription. For example, mutation so as to prevent SUMO modification increases transcription-factor activity associated with Elk-1, Sp3, c-myb, c-jun, AP2, or the p300 N terminus (1-5). In contrast to SUMO, ubiquitin modification is more frequently associated with proteasome-dependent degradation or activation of transcription. In fact, activation of transcription by the VP16 activation domain, in yeast, requires an E3 ubiquitin ligase (6). Addition of a SUMO consensus acceptor site to the Gal4-VP16 activation domain reduces transcription 10-fold (7). Thus, ubiquitin and SUMO modifications can have opposite effects on transcription.Despite these differences, SUMO-1, -2, and -3 are ubiquitinlike proteins and have ubiquitin-like structures. SUMO-2 and -3 are 95% identical and are 46% and 48%, respectively, identical to SUMO-1. In contrast, SUMO-1, -2, and -3 share only 18% identity with ubiquitin (Fig. 1). The differences in primary sequence and their role in the opposing transcriptional properties of SUMO and ubiquitin are being elucidated. SUMO interaction with histone deacetylases has been proposed as one mechanism of repression (1, 8). However, an unbiased directed screen to identify candidate SUMO-associated repressors has not been undertaken.The objective of our experiments was to identify proteins whose association with SUMO-2 depends on SUMO-2 residues required for repression. Such proteins would be candidate SUMO corepressors. We have u...