Role of Q190 of MuLV RT in ddNTP resistance and fidelity of DNA synthesis: a molecular model of interactions with substrates.
Gln190 of MuLV reverse transcriptase (RT) plays an important role in the catalytic mechanism of MuLV RT for its conservative and non-conservative mutant derivatives exhibit low catalytic activity. We now report that both Q190N and Q190A MuLV RTs are more efficient in their activity to incorporate ddNTPs and exhibit higher fidelity than the wild-type (WT) enzyme of DNA synthesis in both RNA- and DNA-directed reactions. To obtain some insight into the structural basis for the differential utilization of dNTP and ddNTP by the mutant enzymes, we modeled the binary and the ternary complexes of MuLV RT using corresponding HIV-1 RT structures and available structure of the fragment of MuLV RT. Q190 of MuLV RT appears to be essential for the interaction with 3'OH of dNTP. The lack of a 3'OH moiety in ddNTP does not permit the binding of ddNTPs to WT MuLV RT. However, the shorter side chain of Q190N (or A) mutant MuLV RT and the absence of 3'OH in ddNTP result in the rearrangement of hydrophobic interactions favoring the binding and limited incorporation of ddNTPs. In addition, while modeling the binary and ternary complexes of MuLV RT, we noted that in the formation of the ternary complex, an interaction of Q190 with dNTP substrate requires a shift from its interaction with the template base. This may be achieved by a small conformational change or motion of the loop between beta9 and alphaH containing Q190, which may correspond to the conformational change step requiring participation of Q190 during the catalytic reaction as reported in an earlier biochemical investigation.
Singh
K
K
Department of Biochemistry and Molecular Biology, UMD-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, NJ 07103, USA.
Kaushik
N
N
Jin
J
J
Madhusudanan
M
M
Modak
M J
MJ
D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003854:Deoxyribonucleotides; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase