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Identification of intrinsic order and disorder in the DNA repair protein XPA.

ABSTRACT: The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

SUBMITTER: Iakoucheva LM

PROVIDER: S-EPMC2374143 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Identification of intrinsic order and disorder in the DNA repair protein XPA.

Iakoucheva L M LM Kimzey A L AL Masselon C D CD Bruce J E JE Garner E C EC Brown C J CJ Dunker A K AK Smith R D RD Ackerman E J EJ

Protein science : a publication of the Protein Society 20010301 3

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolyt ...[more]

PMID: 11344324

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Project description:As an indispensable factor in DNA damage recognition step of nucleotide excision repair, XPA interacts with a series of proteins to initiate repair process. The expression characteristics of XPA in colorectal cancer (CRC) and its influence on CRC prognosis remain elusive. Tissue specimens of CRC and nontumor adjacent tissues from 283 patients were collected. XPA protein expressions were detected by immunohistochemistry staining. Nonparametric test was used to investigate the difference of XPA expression between CRC and nontumor adjacent tissues, as well as the correlation between XPA expression and clinicopathological parameters of CRC. Univariate and multivariate Cox proportional hazards models were applied to estimate the relationship between XPA expression and CRC prognosis. Meanwhile, we analyzed TCGA data to investigate the relation between XPA mRNA expression and survival of CRC. XPA protein expression was significantly decreased in CRC tissues compared with nontumor adjacent tissues (P = 0.001). Subgroup analysis indicated consistently significant down-regulation of XPA in CRC tissues in age > 60 (P = 0.026), age ≤ 60 (P = 0.008), colon cancer (P = 0.009), and rectal cancer (P = 0.015) patients and males (P = 0.004). For clinicopathological parameters, CRC patients with drinking habits revealed XPA overexpression than nondrinkers (P = 0.032). For prognosis, CRC patients with high XPA protein expression had longer overall survival (OS) (HR = 0.62, 95%CI: 0.39-0.97, P = 0.037). Stratified analysis suggested a better prognosis in relation to high XPA protein expression in patients over 60 years (adjusted HR = 0.48, P = 0.021), with rectal cancer (HR = 0.56, P = 0.037), without distant metastasis (HR = 0.58, P = 0.033), without tumor deposits (HR = 0.40, P = 0.006; adjusted HR = 0.44, P = 0.028), and with tumor diameter over 4 cm (HR = 0.49, P = 0.023). DNA repair protein XPA is significantly decreased in colorectal cancer tissues than in adjacent nontumor tissues. High expression of XPA protein showed significant relationship with better survival of CRC, especially rectal cancer. XPA might be a novel biomarker but might not be an independent factor to predict prognosis of CRC patients.

Dataset Information

Identification of intrinsic order and disorder in the DNA repair protein XPA.

Publications

Identification of intrinsic order and disorder in the DNA repair protein XPA.

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