Structural Biochemistry/Polynucleotide Kinase/phosphatase: Its role in DNA repair

Overview
DNA is the essential blueprint of biological life that is required for many important processes such as the transfer of hereditary information from generation to generation, and protein production. DNA repair mechanism that has been developed through evolution works to maintain the integrity of the genome. Specifically, when a mistake is present in the gene, DNA polymerase corrects the mistake and the strands are rejoined by DNA ligases. The polynucleotide kinase/phosphatase plays a crucial role in this process by catalyzing the restoration of 5 prime phosphate and 3 prime hydroxyl termini. PNKP also interacts with other DNA repair proteins such as the XRCC1 and XRCC4 to maintain genomic stability.

Causes of DNA damage and its consequences
There are two main types of DNA damage

1.	Endogenous damage originates from within the cell. Examples of endogenous damage includes attack by reactive oxygen species that is formed as the byproduct of a metabolic process, and replication errors. Specifically, damages include oxidation of bases, alkylation of bases, and hydrolysis of bases.

2.	Exogenous damage is caused by various external factors. Examples include, ultraviolet radiation from the sun, plant toxins, viruses, and mutagenic chemicals. UV light forms pyrimidine dimers and free radicals and breaks in the DNA strand Damages in DNA can lead to uncontrollable cell division and cancer. It can also cause alteration or loss of gene function. This in turn may affect the production of proteins involved in essential enzymatic processes and lead to many other disease. Also, DNA damage such as double strand breaks may trigger cytotoxic effects which has been closely linked with ageing.

Structure of PNKP
PNKP has a N-terminal forkhead associated domain and a C-terminal catalytic domain that is made up of fused phosphatase and kinase subdomains. The forkhead domains adopt a beta sandwich fold and series of loops coming out of this structure provide a peptide binding surface for specific targets. The forkhead associated domain selectively binds to the casein kinase 2 phosphorylated areas in XRCC1 And XRCC4. XRCC1 is a key scaffolding protein in the repair of single strand breaks and XRCC4 is a key scaffolding protein in the repair of double strand breaks. The two catalytic active sites are located on the same side of the PNKP. These interactions between proteins allows for the coordinated regulation among these proteins during the process of DNA repair.

There is a significant structural difference between the mammalian and phage enzymes. The phage PNK DNA binding region is compatible with a single stranded substrates but not double stranded substrates. On the other hand, mammalian enzyme more efficiently phosphorylates the 5 prime hydroxyl termini with gaps or double strand breaks with overhanging ends; single stranded 5 prime termini or blunt ends are not as well phosphorylated. The phosphatase sub domain of the PNKP has a haloacid dehalogenase fold. Mechanism of these enzymes is dependent of MG2+ and proceeds through catalytic aspartate and acylphosphate intermediate. The two channels surrounded by positively charged loops allows access to the phosphatase active site during PNKP’s interaction with 3 prime phosphate ends.

Single strand break repair
The single strand break repair involves multiple enzymes that work together to get the job done. Poly AdP ribose polymerase, XRCC1, AP endonuclease I, and PNKP initially recognizes the damage and works to correct it. Replacing the incorrect nucleotides and reattaching the broken strands can happen by a short patch pathway or a long patch pathway. The short patch pathway involves DNA polymerase beta and NA ligase III and the long patch pathway involves DNA polymerase delta, FEN1 endonuclease and DNA ligase I. The APE1 removes the 3 prime phosphoglycolates while PNKP hydrolyses 3 prime phosphate groups and phosphorylates the 5 prime OH termini. The exact model for the interaction between PNKP, and other proteins such as XRCC1, and CK2 is unknown but more research is being conducted in these areas.

Base excision repair
Most of the minor base errors are fixed through the base excision repair. The incorrect base is first removed by DNA glycosylase and the DNA is cleaved by APE1 at the site where the mistake was present. NEIL1 and NEIL2 are endonucleases that generate 3 prime phosphate termini and also cleave abasic sites. Although these glycosylases doesn’t directly bind to PNKP they interact with PNKP through larger complexes that contain other base excision repair components. Through their cleavage activity, NEIL1 and NEIL2 provide an alternative BER pathway that does not involve dna cleavage by APE1.

Double strand break repair
Experiments suggest that PNKP participates in nonhomologous end joining but does not play a role in homologous recombination. XRCC4 links PNKP to DNA ligase IV and the CK2 mediated phosphorylation of XRCC4 leads PNKP forkhead domain to interact with XRCC4. This entire interaction stimulates the ligation of a 5 prime dephosphorylated plasmid substrate. PNKP works with TDP1 to process double strand break 3 prime phosphoglycolate termini in nonhomologous end joining pathway.

Clinical applications
PNKP is of particular interest to researchers because it is involved in many of the DNA repair pathways that protect cells from endogenous and exogenous genotoxic agents. Microcephaly, seizures, and developmental delay were observed from individuals who had PNKP mutations. High level of PNKP in arthrofibrotic tissue suggests that PNKP mitigates the adverse effects of reactive oxygen species generated by macrophages. All these points demonstrate the clinical importance of PNKP.

In other instances scientists have actually targeted PNKP for cancer therapy. Many cancers up regulate DNA repair enzymes and become resistance to chemotherapy and radiation treatment. Inhibitors of DNA repair pathway can be used to combat this effect to make cancer fighting drugs more effective. In fact, certain PNKP inhibitors have been shown to increase sensitivity of human lung and breast cancer cell to topoisomerase I inhibitors and radiation. Methods of targeting DNA repair pathways, such as the one in which PNKP is involved, has allowed for new ways of fighting cancer.

Conclusion
PNKP works in conjunction with many other proteins to maintain the stability and integrity of the genome. Polynucleotide kinase/phosphatase mediates the DNA repair process by catalyzing the restoration of 5 prime phosphate and 3 prime hydroxyl termini. By studying the structure of PNKP and the specific pathway in which it is involved, scientists have been able to come up with various clinical applications involving PNKP such as new ways to combat cancer.