Increased catalytic activity and altered fidelity of human DNA polymerase ι in the presence of manganese

被引:105
作者
Frank, Ekaterina G. [1 ]
Woodgate, Roger [1 ]
机构
[1] NICHD, Lab Genom Integr, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1074/jbc.M702159200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg2+ is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn2+ in vitro. Here we report on the effects of Mn2+ and Mg2+ on the enzymatic properties of human DNA polymerase iota (pol iota). pol iota exhibited the greatest activity in the presence of low levels of Mn2+ (0.05-0.25 mM). Peak activity in the presence of Mg2+ was observed in the range of 0.1-0.5 mM and was significantly reduced at concentrations > 2 mM. Steady-state kinetic analyses revealed that Mn2+ increases the catalytic activity of pol iota by similar to 30-60,000-fold through a dramatic decrease in the Km value for nucleotide incorporation. Interestingly, whereas pol iota preferentially misinserts G opposite T by a factor of similar to 1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mM Mg2+, respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mM Mn2+. Low levels of Mn2+ also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol iota for Mn2+ even when Mg2+ is present in a > 10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn2+ rather than Mg2+, as tacitly assumed.
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页码:24689 / 24696
页数:8
相关论文
共 48 条
[1]  
ASH DE, 1982, J BIOL CHEM, V257, P9261
[2]   5′-Deoxyribose phosphate lyase activity of human DNA polymerase ι in vitro [J].
Bebenek, K ;
Tissier, A ;
Frank, EG ;
McDonald, JP ;
Prasad, R ;
Wilson, SH ;
Woodgate, R ;
Kunkel, TA .
SCIENCE, 2001, 291 (5511) :2156-2159
[3]   ON THE FIDELITY OF DNA-REPLICATION - MANGANESE MUTAGENESIS INVITRO [J].
BECKMAN, RA ;
MILDVAN, AS ;
LOEB, LA .
BIOCHEMISTRY, 1985, 24 (21) :5810-5817
[4]  
BESSMAN MJ, 1958, J BIOL CHEM, V233, P171
[5]   Human DNA polymerase λ diverged in evolution from DNA polymerase β toward specific Mn++ dependence:: a kinetic and thermodynamic study [J].
Blanca, G ;
Shevelev, I ;
Ramadan, K ;
Villani, G ;
Spadari, S ;
Hübscher, U ;
Maga, G .
BIOCHEMISTRY, 2003, 42 (24) :7467-7476
[6]   Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases [J].
Choi, Jeong-Yun ;
Chowdhury, Goutam ;
Zang, Hong ;
Angel, Karen C. ;
Vu, Choua C. ;
Peterson, Lisa A. ;
Guengerich, F. Peter .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2006, 281 (50) :38244-38256
[7]   Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι [J].
Choi, JY ;
Guengerich, FP .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2006, 281 (18) :12315-12324
[8]  
Creighton S, 1995, METHOD ENZYMOL, V262, P232
[9]   Participation of mouse DNA polymerase ι in strand-biased mutagenic bypass of UV photoproducts and suppression of skin cancer [J].
Dumstorf, Chad A. ;
Clark, Alan B. ;
Lin, Qingcong ;
Kissling, Grace E. ;
Yuan, Tao ;
Kucherlapati, Raju ;
McGregor, W. Glenn ;
Kunkel, Thomas A. .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2006, 103 (48) :18083-18088
[10]   MOLECULAR MECHANISMS OF MANGANESE MUTAGENESIS [J].
ELDEIRY, WS ;
DOWNEY, KM ;
SO, AG .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (23) :7378-7382