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Protein Function

Major Functions of XPA (11)
XPA is a DNA damage recognition and repair factor protein, initiating repair by binding to damaged sites
  1. DNA excision repair
  2. Regulation of autophagy
  3. Protein localization to nucleus
  4. Metal ion binding
  5. Protein homodimerization activity
  6. Response to auditory stimulus
  7. Response to oxidative stress
  8. Response to toxic substance
  9. UV-damage excision repair
  10. UV protection
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Most Common Functions
Figure 1. (Base Excision Repair)
Diagram created by Jeffrey Chimenti
Created using Adobe Photoshop 2o2o
NER3.png
Figure 1: In transcription-coupled repair (TCR), RNA polymerase II (RNAP II) is sited at a lesion. From this point, TCR factors are recruited  to the site, resulting in the RNAP II to be removed in order to allow the transcription factor II H (TFIIH) access along with other nucleotide excision repair (NER) enzymes. During global genome repair (GGR-NER), there is a helix distorting lesion that can be directly recognized by the XPC complex with hRAD23B and centrin 2 (CETN2). (29) Throughout this process, there are lesions that do not significantly destabilize the DNA duplexes. This lack of destabilization is first seen by XPE and DDB1, creating a kink that is recognized by XPC.(29) The DNA is then broken down by the lesion and attracts the TFIIH complex. TCR and GGR then bind allowing XPB and XPD to unwind the DNA and create a bubble of approximately 30 nucleotides. At this point XPA and RPA bind recruiting XPG which releases the XPC complex. XPA is seen to bind at the 5′ side of the bubble, whereas RPA binds to the ssDNA, in the opposite side the lesion. This DNA binding is seen to both protect it from degradation, as well as act as a way to organize base pair excision and DNA repair. (29)
Figure 2. (Damaged DNA Binding)
Diagram created by Jeffrey Chimenti
Created using Adobe Photoshop 2o2o
Bind.png
Figure 2: XPA and RPA both have the ability to form a complex in the absence of DNA, showing that the interaction of XPA with RPA is essential for proper binding. The three part complex of XPA, RPA, and DNA shows that these proteins can either function independently from one another, or together depending on their abundance during different phases of binding to select damaged DNA. Figure 2 shows the approximate locations of RPA and XPA proteins during the pre and post excision complexes. (12) RPA binds to the undamaged DNA strand, which then allows XPA to be recruited into the complex. This interaction of XPA and RPA binding to the DNA site also allows for other proteins involved in NER to bind for re-synthesis. (12) This shows that XPA can be distinguished as a key factor for the completion of the protein complex that is formed on damaged DNA during the pre-incision step as well as the following nucleotide excision repair steps. (12)
Biochemical Pathways
XPA is notably known for two major pathways
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  1. Platinum drug resistance
  2. Nucleotide excision repair
     
Figure 3. Platinum drug resistance (13)
hsa01524.png
Figure 3: Platinum-based drugs such as cisplatin, carboplatin and oxaliplatin are frequently used for therapy of malignant tumors for diseases like testicular, ovarian, colorectal, bladder, and lung cancers. These platinum-based drugs use a specific pathway that involves covalent binding to purines of the DNA bases. (13) The binding of these drugs subsequently lead to cellular apoptosis. These drugs have limitations in context to their success due to the amount of side effects that they cause as well as intrinsic/acquired resistance. This resistance is said to be caused by many different factors including the involvement of XPA and its ability to repair damaged DNA. XPA is seen to bind less to damaged sites of the DNA during the platinum drug resistance pathway and increase DNA repair. This then causes decreased mismatched pairs and defective apoptosis. (13)
Figure 4. Nucleotide Excision Repair (14)
hsa03420.png
Figure 4: Nucleotide excision repair (NER) is a pathway involving XPA that is used to diagnose and repair bulky DNA damage caused by various different factors such as environmental carcinogens, and exposure to UV-light. Many types of hereditary diseases involving the NER pathway affect humans such as Cockayne syndrome, trichothiodystrophy, and the XPA involvement for xeroderma pigmentosum. (14) Repair of damaged DNA in the NER mechanism are known to involve about 30 polypeptides with two different sub-pathways. These two sub-pathways are called transcription-coupled repair (TCR-NER) and global genome repair (GGR-NER). (15) Transcription-coupled repair is involved with the repair of lesions in the actively transcribed strand of genes by RNA polymerase II (RNAP II). Global genome repair NER is involved with XPA and its ability to recognize, bind, and repair DNA that is damaged. GGR-NER also uses the XPC-hHR23B complex in conjunction with the XPE complex (14). XPA is then brought to the damaged site by the transcription factor II H (TFIIH) complex that will then unwind the double stranded DNA around the damaged nucleotide creating the NER bubble. (15) XPA also does this while simultaneously binding to the ssDNA binding protein replication protein A (RPA). Both XPA and RPA help recruit and properly position the excision nucleases for proper mechanistic progression. (15)
Tissue Expression (16)
XPA is expressed throughout each of its isoforms in the following tissues:
  • Brain
  • Heart
  • Skeletal muscle
  • Colon
  • Kidney
  • Liver
  • Lung
  • Thyroid
  • Adrenal gland
  • Breast
  • Ovary
  • Prostate
  • Testis
  • Adipocyte
  • Spleen
  • Esophagus
  • Bladder
  • Pancreas
  • Salivary gland
  • Pituitary
XPA is ubiquitous and is expressed throughout all of its isoforms (16)
Developmental Stages (17)
XPA is expressed in all stages of the human life cycle (17):

  • Late embryonic stage

  • Infant stage

  • Adolescent stage

  • Young adult stage

  • Human middle aged stage

  • Human late adult stage

Sub-Cellular Localization (18)
Figure 5. XPA Sub-Cellular Localization (18)
Screen Shot 2019-12-02 at 2.22.20 PM.png

The sub-cellular localization relates to the location inside of the cell that the protein is present in or functions in. Figure 5 shows a diagram that is color coordinated in relation to the level of confidence of the expressed organelles.

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In context of isoforms 'a' & 'b', XPA is mainly localized within the nucleus, as well as the extracellular space. XPA can also be found within the cytosol, and is less likely to be found within the golgi apparatus.

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Expression in variant a: 47% in nucleus, 30% in mitochondria, 13% in cytoplasm, 8% in cytoskeleton. (3)

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Expression in variant b: 43% in cytoplasm, 21% in mitochondria, 17% in nucleus, 8% in cytoskeleton, 4% in golgi apparatus, 4% in peroxisomes. (3)

Sub-cellular localization has a large impact on the function of XPA because it is a complementing protein that is heavily involved in DNA excision repair that must be present in organelles such as the nucleus. XPA recognizes and binds to damaged DNA for repair, signifying that its sole task must be accomplished inside of the nucleus for both of its isoforms. (11)
Figure 6. Top Five Protein Interactions (18)
Screen Shot 2019-12-02 at 2.25.02 PM.png
XPA interacts with many different proteins, but the five most common are AQR, POLE2, RPA3, RBX1, and PIAS1 shown in figure 6. (18)
 
AQR- Involved in pre-mRNA splicing as a component of the spliceosome. Intron-binding spliceosomal protein that aids in linking pre-mRNA splicing and snoRNP biogenesis. Localized with XPA within the nucleus sharing similar binding properties. (19)
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POLE2- This is an accessory component of the DNA polymerase epsilon complex. Participates in DNA repair and in chromosomal DNA replication similar to XPA’s DNA repair function located within the nucleus. (20)
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RPA3- This replication protein binds and stabilizes single stranded DNA intermediates that form during DNA replication or when there is DNA stress. It plays an important role in both DNA replication and the cellular response to DNA damage. It is localized within the nucleus of the cell with XPA, sharing similar biological processes such as base-excision repair, and binding to damaged DNA. (21)
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RBX1- This E3 ubiquitin-protein ligase is apart of complexes that mediates the ubiquitination and following proteasomal degradation of target proteins such as proteins involved in cell cycle progression, signal transduction, transcription and transcription-coupled nucleotide excision repair. It is localized within the nucleus mainly as well as the cytoplasm. It also shares multiple biological processes with the XPA protein such as DNA damage response, detection of DNA damage, and genome nucleotide excision repair. (22)
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PIAS1- This protein functions as an E3 SUMO-protein ligase, working to stabilize the interaction between UBE2I and the substrate, and as a SUMO-tethering factor. It is localized within the nucleus and shares the molecular function of DNA binding with the XPA gene. (23)
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