The XPA Protein-Life under Precise Control

Abstract

Nucleotide excision repair (NER) is a central DNA repair pathway responsible for removing a wide variety of DNA-distorting lesions from the genome. The highly choreographed cascade of core NER reactions requires more than 30 polypeptides. The xeroderma pigmentosum group A (XPA) protein plays an essential role in the NER process. XPA interacts with almost all NER participants and organizes the correct NER repair complex. In the absence of XPA's scaffolding function, no repair process occurs. In this review, we briefly summarize our current knowledge about the XPA protein structure and analyze the formation of contact with its protein partners during NER complex assembling. We focus on different ways of regulation of the XPA protein's activity and expression and pay special attention to the network of post-translational modifications. We also discuss the data that is not in line with the currently accepted hypothesis about the functioning of the XPA protein.

Keywords: ATR; DNA repair; PARP1; PARylation; XPA; nucleotide excision repair (NER); phosphorylation; post-translational modifications.

Figure 1

XPA’s structure and interaction partners. (A) The map of the XPA domain structure and known points of PTMs: phosphorylation at S196 and acetylation at K63 and K67. Secondary-structure elements are shown according to crystal structures PDB 6LAE and 6J44: β-strands are green (β1: aa 103–104, β2: aa 111–112, β3: aa 138–140, β4: aa 164–167, and β5: aa 178–172), and α-helices are light blue (α1: aa 116–121, α2: aa 141–148, α3: aa 151–157, α4: aa 183–194, and α5: aa 197–239). Positively charged residues K141, K151, K179, R207, and R211, which are directly involved in interactions with backbones of a DNA duplex, are shown as blue triangles. Two residues (Thr140 and Thr142, indicated as purple triangles) interact with the DNA backbone through a van der Waals contact and a hydrogen bond, respectively. Extended helix α5 contains several positively charged residues (Lys217/218/221/222/224/236 and Arg227/228/231/237) that are possibly involved in DNA binding, which are shown as a blue striped box. Conserved residue Trp175 intercalates into unpaired bases of single-stranded DNA (ssDNA) at the ss–dsDNA junction and is displayed as a red circle. Unstructured N- and C-terminal regions are gray. Zinc-coordinated conserved cysteine residues (C105, C108, C126, and C129) are presented as red asterisks and a Zn-finger motif (ZnF, aa 102–129) colored green. The N terminus accommodates a nuclear localization signal (NLS, aa 30–42), which is yellow. DNA-binding (aa 98–239) and poly(ADP-ribose) (PAR)-binding (aa 213–237) motifs are mapped to the overall XPA structure and are highlighted in dark blue. (B) Interaction sites for NER protein partners on XPA, which are aligned with the XPA residues involved in each interaction. Proteins whose interaction sites are unknown are gray. (C) XPA interaction partners outside NER. (D) A structural model of the XPA globular core domain (PDB ID: 6LAE). A ribbon diagram with color codes according to (A). The Trp175 residue is shown in red. A distribution of the electrostatic potential on the surface for the same structure: a positive charge is shown in blue, and a negative charge is red. The structures were generated using UCSF Chimera software (version 1.16).

Figure 2

XPA interaction with the ss–dsDNA junction. (A) Cryo-EM structure PDB: 6RO4 provides details of the XPA–DBD interaction with the ss–dsDNA junction. XPA demarcates the 5′ edge of the DNA repair bubble. XPA inserts its intercalating β-hairpin between DNA single strands at the junction. Red colored Trp175 from the tip of the β-hairpin stacks against the base of the DNA 3′-extension at the junction. The structures were generated using UCSF Chimera software (version 1.16). (B) Schematic representation of the interactions between side chains of the XPA and DNA junction, according to cryo-EM structure PDB: 6RO4 [21] and crystal structure PDB: 6LAE. DNA nucleotides are indicated as circles. Patches of positively charged residues in proximity to the DNA backbone are indicated by red pluses. Hydrogen bonding of T142 and a van der Waals contact of T140 are indicated as black lines.

Figure 3

XPA organizes GG-NER machinery functioning. (Initial damage recognition) XPA interacts physically with DDB2 and XPC, but the biological role of these interactions is not understood. (Damage verification) XPC recruits TFIIH through XPC–p62 interaction. When XPA joins this complex through interaction with p8 and p52, it induces TFIIH rearrangement through binding XPB. During XPB binding, XPA engages the Cen2-binding site on XPB and possibly promotes XPC complex displacing. Next, XPG joins the complex by occupying the CAK-binding site on XPD. Thus, XPA and XPG promote CAK releasing and XPA “turning on” TFIIH repair conformation. During the damage verification, XPA retains XPB on DNA (see also Figure 4), modulates XPD helicase activity, and further inhibits it in the presence of a bulky lesion. (Pre-incision complex) XPA drives RPA binding to the undamaged strand through XPA–RPA32 interaction and stabilizes the PIC interior by additional interaction with RPA70. PIC becomes completed after XPF–ERCC1 engagement, which is recruited by XPA. XPA is a central component in the PIC, which makes sure that all the NER factors are in the right place for the incision to occur. At the later stages, XPA remains in the NER complex via a protein–protein interaction with the RPA that stays bound to the undamaged strand. Additionally, XPA possibly promotes the positioning of the PCNA clamp.

Figure 4

Cryo-EM structure of the human core TFIIH–XPA–DNA complex (PDB ID: 6RO4). XPA is colored light blue. The TFIIH subunits that are directly involved in the interaction with XPA: XPB, XPD, p52, and TTDA/p8 are colored yellow, magenta, green, and orange, respectively. In this complex, XPA forms a bridge between XPB and XPD, and XPA’s extended α5 helix and XPB form a positively charged tunnel that holds the DNA duplex within. XPA binds to XPD via its intercalating β-hairpin. The C-terminal region of XPA should be extended to p52 and TTDA/p8 and bind to these subunits, but unfortunately, it was observed at a lower resolution and was not used for the final model building (for details, please, see original research article [21]). The structures were generated using UCSF Chimera software (version 1.16).

Figure 5

The circadian rhythm of XPA’s life. (A) The circadian rhythm of NER activity is due to the circadian oscillation of XPA mRNA and the protein levels. In a mouse model, it was shown for the brain, skin, and liver that the elimination of cisplatin–DNA adducts by the NER system exhibits a robust circadian rhythm, with the zenith in the late afternoon hours (~5 p.m.) and the nadir in the early morning hours (~5 a.m.). This oscillation is caused by the circadian rhythmicity of Xpa transcription and translation. (B) The balance between production and degradation. The mammalian circadian clock is generated by a transcriptional–translational feedback loop: core clock proteins CLOCK and BMAL1 activate the transcription of many clock-controlled genes, including repressor genes period (PER1/2) and cryptochrome (CRY1/2), by binding to E-box elements in their promoters and activating their transcription. After a time delay, the CRY and PER proteins accumulate in the cytoplasm, then form the CRY–PER complex, and are translocated back into the nucleus to inhibit their own transcription, as well as the transcription of clock-controlled output genes, through the inhibition of CLOCK–BMAL1 activity. The amount of repressor proteins CRY and PER is also regulated by secondary feedback loops at the transcriptional level, as well as by proteolytic degradation [126]. Accordingly, the XPA mRNA and protein levels are regulated positively by the CLOCK–BMAL1 complex and negatively by the CRY–PER complex. To attain a sufficient amplitude of the XPA protein oscillation level, circadian transcriptional regulation is coupled with the permanent prompt removal of the XPA protein by ubiquitin-dependent degradation. (C) UV light-induced damage and protective phosphorylation. In response to UV in a dose-dependent manner, ATR binds to and phosphorylates XPA. The XPA–ATR interaction facilitates HERC2 dissociation from the XPA complex, resulting in the accumulation of XPA molecules.

Figure 6

The ATR interaction site on XPA is the α4-helix located on the N-terminal side of the α4-helix-turn-α5-helix motif. ATR phosphorylates XPA at serine 196 (S196), which is located in the “turn” element inside the helix-turn-helix motif. The color codes correspond to Figure 1A. The structure was generated using UCSF Chimera software (version 1.16).

Figure 7

The network of reciprocal regulatory mechanisms of XPA’s PTMs. (A) The putative phosphorylation complex. ATR phosphorylates XPA at S196. (B) Acetylation–phosphorylation regulation. XPA is acetylated at Lys63 and Lys67. SIRT1 promotes NER by deacetylating XPA. The existence of an ATR–SIRT1 interaction is unclear and is indicated by the question mark. (C) PARylation–acetylation interplay. XPA is PARylated by PARP1. Both PARP1 and SIRT1 use NAD⁺ for their activity and interact physically. There is a strong connection between acetylation and PARylation. SIRT1 may deacetylase PARP1 and inhibit the PARP1 enzymatic activity. Under severe stress, PARP1 can become overactivated and may deplete cellular NAD⁺, thereby leading to the repression of SIRT1 activity and suppressing SIRT1 transcription. To prevent this situation, SIRT1 is also capable of negatively regulating the expression of the PARP1 gene. Moreover, XPA has a high affinity for long PAR polymers. Whether the PARylation events involve phosphorylated XPA is unclear and is denoted by the question mark.