Identification of regulatory factor X as a novel mismatch repair stimulatory factor.
This report describes the identification and purification of a novel mismatch repair stimulatory factor from HeLa cell extracts. This activity copurifies with a proliferating cell nuclear antigen-dependent 5 ' --> 3 ' DNA excision activity during several purification steps but is resolved from the excision activity during gel filtration chromatography using Sephacryl S-300. After purification to near homogeneity, the stimulatory factor is associated with three polypeptides with apparent molecular masses of 68, 36, and 30 kDa. Peptide sequencing analysis by tandem mass spectrometry identified the stimulatory factor as the heterotrimeric regulatory factor X (RFX) complex, which regulates transcription of the class II major histocompatibility complex by facilitating histone acetylation and is defective in the human hereditary immunodeficiency syndrome called bare lymphocyte syndrome. This conclusion was confirmed by the facts that purified recombinant RFX stimulates mismatch repair in an in vitro reconstituted mismatch repair system and that depletion of RFX from nuclear extracts or RFX knockdown in cells reduces mismatch repair activity. As expected, RFX knockdown cells display instability in microsatellite sequences. The possible role of RFX in human MMR repair is discussed.
Zhang
Yanbin
Y
Department of Toxicology, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Yuan
Fenghua
F
Wang
Daojing
D
Gu
Liya
L
Li
Guo-Min
GM
D002384:Catalysis; D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C093524:RFX5 protein, human; D034741:RNA, Small Interfering; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors