Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase α and Klenow Fragment

Abstract

We studied the incorporation of the fluorescent cytidine analogues 1, 3-diaza-2-oxo-phenothiazine (tC) and 1, 3-diaza-2-oxo-phenoxazine (tCo) by human DNA polymerase α and Klenow fragment of DNA polymerase I (E. coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly size expanded toward the major groove. Despite the size alteration both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only ∼4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs, and can incorporate at least 4 consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. KF inserts dGTP with a 4-to 9-fold higher probability than dATP, while pol α favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as templating base and as incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o).

Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

“…DinB synthesizes DNA up to and across from tC, accurately incorporating G, but cannot efficiently bypass tC. It has also been observed that tC can tautomerize to a form that base pairs with A; 41,42 however, similar to human DNA primase and RNA polymerase, 42,43 we did not observe misincorporation by DinB. The tC analog is known to be bypassed by the A-family polymerase Klenow fragment, as well as by human DNA polymerase α primase.…”

“…The tC analog is known to be bypassed by the A-family polymerase Klenow fragment, as well as by human DNA polymerase α primase. 28,41 Specifically, the Klenow fragment accurately inserts dGTP opposite template tC 28 and can then extend the primer to the end of the template. 41 Remarkably, the Klenow fragment is also fairly proficient at bypassing 8-oxo-G and C 8 -aminofluorene-dG.…”

“…In contrast, both Klenow fragment and human DNA polymerase α can insert dGTP opposite template tC and extend past the noncanonical base pair to the end of the template. 28,41 This is an intriguing difference, as replicative polymerases are generally considered to be accurate and specific for canonical base pairs, 11 whereas Y-family polymerases have more open and solvent-accessible active sites in order to accommodate bulky lesions in DNA. 2,6,9 Results DinB discriminates against tC as the template base…”

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III

“…First, a short turn connecting ␣-helices ␣B and ␣C, together with the adjacent helix parts (Ser 40 -Leu 47 ), is inserted into the minor groove of the DNA double helix to push the damaged nucleotide out. The void left after the eversion is filled by Gln 42 , and the adjacent bases are stacked against its side chain amide group (the 3Ј-base) and the planar peptide bond Gln This arrangement of DNA and protein residues provides insights into the conformational transition dynamics associated with the changes in fluorescence of the reporters incorporated into DNA (Fig. 8).…”

“…The oxoG base is then fully placed into the active site and forms specific interactions within the base recognition pocket detected in the structure of the pre-catalytic hOGG1-DNA complex (22). In addition, Asn 149 (corresponding to Gln 42 in G. stearothermophilus Endo III, Fig. 3B) forms a hydrogen bond between the amide carbonyl of its side chain and the amino group of the opposite cytosine.…”