In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.
In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta.
复制标题
酵母 DNA 聚合酶 α、ε、δ 和 zeta 中假定的活性位点突变的体内后果。
DOI：
10.1093/genetics/159.1.47
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发表时间：
2001
期刊：
影响因子：
3.3
通讯作者：
Kunkel,TA
中科院分区：
文献类型：
--
作者：
Pavlov,YI;Shcherbakova,PV;Kunkel,TA
A家族DNA聚合酶活性部位的几个氨基酸对DNA的准确合成起着重要作用。对于其中两个残基，B家族DNA聚合酶在II和III区域保守酪氨酸残基，这两个区域被认为具有类似的功能。在这里，我们用酵母DNA聚合酶α，δ，ε和ζ的催化亚基中的丙氨酸取代了每个酪氨酸，并检测了其在活体中的结果。在POLδ或POLε中，与保守的SL/MYPS/N基序中的酪氨酸取代的株是不可行的。在POLζ的催化亚基Rev3中具有相同替换的菌株几乎是紫外线不变的，这表明功能严重丧失。在POLα(Pol1-Y869A)中有这种替换的菌株是活的，但它表现出生长缓慢，对羟基脲敏感，并且对移码和碱基替换具有自发的突变表型。Pol1-Y869A/pol1-Y869二倍体表现出异常生长。因此，这种酪氨酸对所有四种真核生物B家族DNA聚合酶的功能都是至关重要的。在POLα，-δ，或-ε中，在保守的NS/VxYG基序中有酪氨酸替换的菌株是活的，而在Rev3中有同源替换的菌株是紫外线突变的。POLα突变体没有明显的表型。Polε(Pol2-Y831a)突变体对羟基脲轻微敏感，是自发碱基替换和移码的半显性突变体。Polδ突变体(Pol3-Y708A)生长缓慢，对羟基脲和甲烷磺酸甲酯敏感，是一种强碱基取代和移码突变体。Pol3-Y708A/pol3-Y708二倍体生长缓慢且异常。通过失活依赖于PMS1的α，-δ错配修复，POLε和-PMS1突变株的突变率以位点特异性的方式增加，这表明突变效应是由于染色体DNA复制的保真度降低所致。这可能是由于聚合酶活性部位的氨基酸变化导致突变聚合酶对碱基的选择性松弛直接造成的。此外，丙氨酸取代可能会削弱催化功能，使不同的聚合酶在复制分叉上竞争。观察到pol3-Y708突变是隐性的，其突变效应被REV3基因的破坏部分抑制，这支持了这一点。
Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases α, δ, ε, and ζ and examined the consequencesin vivo.Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Polδ or Polε are inviable. Strains with same substitution in Rev3, the catalytic subunit of Polζ, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Polα (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. Thepol1-Y869A/pol1-Y869Adiploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Polα, -δ, or -ε are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Polα mutant has no obvious phenotype. The Polε (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Polδ mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. Thepol3-Y708A/pol3-Y708Adiploid grows slowly and aberrantly. Mutation rates in the Polα, -δ, and -ε mutant strains are increased in a locus-specific manner by inactivation ofPMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that thepol3-Y708Amutation is recessive and its mutator effect is partially suppressed by disruption of theREV3gene.