Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions (original) (raw)
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Isolation and characterization of HC1: a novel human DNA repair gene
Genetics and Molecular Research, 2009
Evolution has selected several systems to protect the genetic material from damage by environmental agents or by products of metabolism, thus preventing the accumulation of mutations or cell death. These systems, which are DNA repair and lesion tolerance mechanisms, exist in all living organisms to maintain genetic stability. The vigorous efforts to isolate and characterize DNA repair genes have helped to understand some aspects of the cell mechanisms for DNA damage processing and their involvement with human syndromes and cancer development . The complexity of DNA repair mechanisms is far from being elucidated, and certainly a larger number of genes are involved in nuclear genome stability. The isolation and characterization of several related genes contribute not only to our knowledge of the cell and gene metabolism, but also help in the prevention of cancer, and this study has revealed an important degree of homology and analogy even in phylogenetically unrelated species .
HMGB1: The jack-of-all-trades protein is a master DNA repair mechanic
Molecular Carcinogenesis, 2009
The high mobility group protein B1 (HMGB1) is a highly abundant protein with roles in several cellular processes, including chromatin structure and transcriptional regulation, as well as an extracellular role in inflammation. HMGB1's most thoroughly defined function is as a protein capable of binding specifically to distorted and damaged DNA, and its ability to induce further bending in the DNA once it is bound. This characteristic in part mediates its function in chromatin structure (binding to the linker region of nucleosomal DNA and increasing the instability of the nucleosome structure) as well as transcription (bending promoter DNA to enhance the interaction of transcription factors), but the functional consequences of HMGB1's binding to damaged DNA is still an area of active investigation. In this review we describe HMGB1's actions in the nucleotide excision repair (NER) pathway, and we discuss aspects of both the "repair shielding" and "repair enhancing" hypotheses that have been suggested. We also report information regarding HMGB1's roles in the mismatch repair (MMR), non-homologous end-joining (NHEJ), and V(D)J recombination pathways, as well as its newly-discovered involvement in the base excision repair (BER) pathway. We further explore the potential of HMGB1 in DNA repair in the context of chromatin. The elucidation of HMGB1's role in DNA repair is critical for the complete understanding of HMGB1's intracellular functions, which is particularly relevant in the context of anti-HMGB1 therapies that are being developed to treat inflammatory diseases.
Nucleic Acids Research, 2007
Growing evidence suggests that the Rad9-Rad1-Hus1 complex (the 9-1-1 complex), besides its functions in DNA damage sensing and signaling pathways, plays also a direct role in various DNA repair processes. Recent studies have demonstrated that the 9-1-1 complex physically and functionally interacts with several components of the base excision repair (BER) machinery namely DNA polymerase b (Pol b), flap endonuclease 1 (Fen 1), DNA ligase I (Lig I) and the MutY homologue of Schizosaccharomyces pombe. In this work, we found for the first time that the 9-1-1 complex interacts in vitro and in vivo with the apurinic/apyrimidinic endonuclease 1 (APE 1), an early component of BER, and can stimulate its AP-endonuclease activity. Moreover, we show that the 9-1-1 complex possesses a stimulatory effect on long patch base excision repair (LP-BER) reconstituted in vitro. The enhancement of LP-BER activity is due to the specific stimulation of the two early components of the repair machinery, namely APE 1 and Pol b, suggesting a hierarchy of interactions between the 9-1-1 complex and the BER proteins acting in the repairosome. Overall, our results indicate that the 9-1-1 complex is directly involved in LP-BER, thus providing a possible link between DNA damage checkpoints and BER.
Journal of Biological Chemistry, 2009
Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase  (pol ), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol  is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol  or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol  with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a "hit and run" mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process. . 3 The abbreviations used are: BER, base excision repair; LP-BER, long patch-BER; nt, nucleotide(s); pol , DNA polymerase ; FEN1, flap endonuclease 1; AP, apurinic/apyrimidinic; APE1, AP endonuclease 1; 9-1-1, Rad 9-Rad1-Hus1; Lig1, DNA ligase 1; dRP, deoxyribose phosphate; DTT, dithiothreitol; BSA, bovine serum albumin; THF, tetrahydrofuran; EMSA, electrophoretic mobility shift assay; PCNA, proliferating cell nuclear antigen; IP, immunoprecipitation.
BMC Molecular Biology, 2014
Background: Human MutY glycosylase homolog (hMYH), a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. Rad9-Rad1-Hus1 (9-1-1) is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and DNA repair. In humans, hMYH and 9-1-1 interact through Hus1 and to a lesser degree with Rad1 in the presence of DNA damage. In Saccharomyces pombe, each component of the 9-1-1 complex interacts directly with SpMYH. The glycosylase activity of hMYH is stimulated by Hus1 and the 9-1-1 complex and enhanced by DNA damage treatment. Cells respond to different stress conditions in different manners. Therefore, we investigated whether Rad9 interacted with hMYH under different stresses. Here, we identified and visualized the interaction between hRad9 and hMYH and investigated the functional consequences of this interaction. Results: Co-IP and BiFC indicates that hMYH interacts with hRad9. As shown by GST-pull down assay, this interaction is direct. Furthermore, BiFC with deletion mutants of hMYH showed that hRad9 interacts with N-terminal region of hMYH. The interaction was enhanced by hydroxyurea (HU) treatment. mRNA and protein levels of hMYH and hRad9 were increased following HU treatment. A marked increase in p-Chk1 (S345) and p-Cdk2 (T14, Y15) was observed. But this phosphorylation decreased in siMYH-or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells. Conclusions: Our data reveal that hRad9 interacts directly with N-terminal region of hMYH. This interaction is enhanced by HU treatment. Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation. Since both protein functions in the early detection of DNA damage, we suggest that this interaction occurs early in DNA damage pathway.
Repair of Hydantoin Lesions and Their Amine Adducts in DNA by Base and Nucleotide Excision Repair
Journal of the American Chemical Society, 2013
An important feature of the common DNA oxidation product 8;oxo;7,8;dihydroguanine (OG) is its susceptibility to further oxidation to produce guanidinohydantion (Gh) and spiroiminodihydantoin (Sp) lesions. In the presence of amines, G or OG oxidation produces hydantoin amine adducts. Such adducts may form in cells via interception of oxidized intermediates by protein;derived nucleophiles or naturally occurring amines that are tightly associated with DNA. Gh and Sp are known to be substrates for base excision repair (BER) glycosylases; however, large Sp;amine adducts would be expected to be more readily repaired by nucleotide excision repair (NER). A series of Sp adducts differing in size of the attached amine were synthesized to evaluate the relative processing by NER and BER. The UvrABC complex excised Gh, Sp and the Sp;amine adducts from duplex DNA, with the greatest efficiency for the largest Sp;amine adducts. The affinity of UvrA with all of the lesion duplexes was found to be similar, whereas the efficiency of UvrB loading tracked with the efficiency of UvrABC excision. In contrast, the human BER glycosylase NEIL1 exhibited robust activity for all Sp;amine adducts irrespective of size. These studies suggest that both NER and BER pathways mediate repair of a diverse set of hydantoin lesions in cells.
Nucleic Acids Research, 1995
HAP1 protein, the major apurinic/apyrimldinic (AP) endonuclease in human cells, Is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichla coliexonuclease III and Drosophlla Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA-and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidlne residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanlne) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA-and RNA-specific nuclease activities of the HAP1 protein.
DNA Repair, 2007
d n a r e p a i r 6 ( 2 0 0 7 ) 410-428 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / d n a r e p a i r Base excision repair pathway Damage reversal Genome maintenance Crystallography Enzyme-DNA complex Structure-specific nuclease Damage recognition Base excision Glycosylase N-glycosylase/AP-lyase enzymes Polymerase FEN-1 PCNA Ligase a b s t r a c t Three-dimensional structures of DNA N-glycosylases and N-glycosylase/apyrimidine/ apurine (AP)-lyase enzymes and other critical components of base excision repair (BER)
HMGB1 Is a Cofactor in Mammalian Base Excision Repair
Molecular Cell, 2007
Deoxyribose phosphate (dRP) removal by DNA polymerase β(Pol β) is a pivotal step in base excision repair (BER). To identify novel BER co-factors, especially those with dRP lyase activity, we used a Pol β null cell extract and BER intermediate as bait for sodium borohydride cross-linking. Mass spectrometry identified the high mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Co-immunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1 −/− mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER co-factor capable of modulating BER capacity in cells.
Coordination of DNA Base Excision Repair by Protein-Protein Interactions
DNA Repair- An Update, 2018
The system of base excision repair (BER) evolved to correct the most abundant DNA damages in mammalian cells is the most essential for maintaining the genome integrity. The multistep BER process involves several enzymes and protein factors functioning in a coordinated fashion that ensures the repair efficiency. The coordination is facilitated by the formation of protein complexes stabilized via either direct or indirect DNA-mediated interactions. This review focuses on direct interactions of proteins participating in BER with each other and with noncanonical factors found recently to modulate the efficiency of BER. All the known partners of main BER participants, the sites responsible for their interaction, and the characteristics of protein-protein affinity are summarized. Well-documented evidences of how DNA intermediates and posttranslational modifications of proteins modulate protein-protein interactions are presented. The available data allow to suggest that the multiprotein complexes are assembled with the involvement of a scaffold protein XRCC1 and poly(ADP-ribose) polymerase 1, a key regulator of the BER process, irrespective of the DNA damage; the composition and the structure of the complexes are dynamically changed depending on the DNA damage, its chromatin environment, and the step of BER process.