PNKP kinase family, and includes both DNA and

PNKP is essential for maintaining genomic
stability. To date, PNKP is the only DNA repair enzyme that has been identified
as possessing 5?-kinase activity (59). In addition, the 3?-phosphatase activity of
PNKP supersedes that of APE1 and aprataxin (APTX), which have been identified
as being able to dephosphorylate 3?-phosphate (59). 
Previous studies revealed major differences in substrate preference
between the kinase and phosphatase domains, and it also showed that both
function independently of one another, with the phosphatase having a faster
catalytic rate (103). The kinase domain, which is selective for
DNA, preferentially phosphorylates nicks, small gaps and recessed 5?-hydroxyl
ends with a 3?-overhang (3, 98, 104). Furthermore, the kinase domain can bind to
double-stranded 5?-termini without base pair disruption (105). This domain belongs to the adenylate kinase family, and includes
both DNA and ATP binding sites. The ATP binding site consists of Walker A
(P-loop) and B motifs (98,
106, 107). The Walker A motif binds the ?- and
?-phosphates of ATP, while the Walker B motif coordinates Mg2+ ion.
The DNA end is sequestered in a pocket of the protein with access to the 5?-OH
terminus and the reaction is catalyzed by the Asp397 residue (Figure 1.5A) (108).

The phosphatase
domain belongs to the haloacid dehalogenase (HAD) superfamily and
contains a conserved DxDGT motif (93,
109). This domain is dependent on Mg2+, which
stabilizes the negative charge on the substrate phosphate during catalysis, and
catalyzes the removal of 3?-phosphate of DNA (110).  The
phosphatase domain utilizes a two-step mechanism, the substrate phosphate is
first held in place for nucleophilic attack by the Asp171 carboxylate to
generate the covalent phospho-aspartate intermediate. In the second step, the
phospho-aspartate is hydrolyzed by Asp172, which deprotonates the attacking
water molecule (93, 110). The dephosphorylation process acts in the
same manner on nicked and gapped
double-stranded substrates and single-stranded substrates as small as 3
nucleotides (86, 93, 105, 108, 110). However, the binding of double-stranded DNA to the PNKP phosphatase domain destabilizes base pairing in the two or three
terminal base pairs of double-stranded substrates closest to the 3?-phosphate
by electrostatic interactions between a positively charged surface of PNKP and
the DNA strand (105).

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PNKP kinase and
phosphatase activities are active on DNA and inactive on RNA.  Mammalian cells have distinct DNA-specific
and RNA-specific polynucleotide kinase activities. The mammalian DNA kinase has
a pH optimum of 5.5, while the RNA kinase has an alkaline pH optimum (111-114). Moreover, the human RNA kinase does not have
an associated 3?-phosphatase, whereas DNA kinases do have an inherent
DNA-specific 3?-phosphatase function (115, 116).