4.2.99.B1 D256A site-directed mutagenesis, C-terminal polymerase domain, completely eliminates dRP lyase activity, proficient in DNA synthesis by the polymerase reaction 694332 4.2.99.B1 D829N/E830Q the polymerase inactive mutant D829N/E830Q retains full 5'-dRP lyase activity. Domain mapping of the 98-kDa enzyme reveals that the 5'-dRP lyase active site resides in a 24-kDa-domain 694467 4.2.99.B1 E71Q retains wild-type enzyme activity 692940 4.2.99.B1 E75A about 75% of wild-type enzyme activity 692940 4.2.99.B1 F25W about 85% of wild-type enzyme activity 692940 4.2.99.B1 H34G about 45% of wild-type enzyme activity 692940 4.2.99.B1 K310A eliminates more than 90% of the wild-type dRP lyase activity, indicating that Lys 310 is the main nucleophile involved in the reaction, forming the Schiff base internediate during beta-elimination 692957 4.2.99.B1 K35A about 50% of wild-type enzyme activity, Lys72 and Lys35 are involved in the dRP lyase reaction catalyzed by the 8-kDa domain and Lys35 is involved in 5'-phosphate recognition. Lys35 may stabilize the leaving group in the dRP lyase reaction through its interaction with the 5'-phosphate of the DNA-terminus 692940 4.2.99.B1 K35A part of the active site, 50% loss of enzyme activity 692010 4.2.99.B1 K35A/K68A part of the active site, similar to wild-type enzyme activity 692010 4.2.99.B1 K35A/K68A/K72A devoid of dRP lyase, Lys72 and Lys35 are involved in the dRP lyase reaction catalyzed by the 8-kDa domain and Lys35 is involved in 5'-phosphate recognition. Lys35 may stabilize the leaving group in the dRP lyase reaction through its interaction with the 5'-phosphate of the DNA-terminus 692940 4.2.99.B1 K35A/K68A/K72A part of the active site, above 95% loss of enzyme activity 692010 4.2.99.B1 K35A/K68A/K72A site-directed mutagenesis, amino-terminal dRP lyase domain, completely eliminates dRP lyase activity, proficient in DNA synthesis by the polymerase reaction 694332 4.2.99.B1 K35A/K68A/K72A/D256A site-directed mutagenesis, amino-terminal dRP lyase domain, deficient in dRP lyase and polymerase activity 694332 4.2.99.B1 K35A/K72A part of the active site, above 95% loss of enzyme activity 692010 4.2.99.B1 K35R part of the active site, no effect on enzyme activity 692010 4.2.99.B1 K35R/K68R/K72R part of the active site, above 95% loss of enzyme activity 692010 4.2.99.B1 K552A site-directed mutagenesis matching the active site of polbeta. The mutant Trf4K552A is overexpressed in a trf4DELTA mutant. The overproduction of Trf4 wild-type increases methylmethane sulfonate resistance similar to that of of the wild-type. The Trf4K552A mutant exhibits hypersensitivity to methylmethane sulfonate, causing double-stranded DNA breaks 692020 4.2.99.B1 K60A about 40% of wild-type enzyme activity 692940 4.2.99.B1 K68A mutation has no significant effect on the removal of 5'-AMP-deoxyribose phosphate 730454 4.2.99.B1 K68A part of the active site, stimulation of enzyme activity 692010 4.2.99.B1 K68A retains wild-type enzyme activity 692940 4.2.99.B1 K68A/E71Q retains wild-type enzyme activity 692940 4.2.99.B1 K68A/K72A about 10% of wild-type enzyme activity 692940 4.2.99.B1 K68R part of the active site, significant reduction of enzyme activity 692010 4.2.99.B1 K72A 2 mM pyridoxal 5'-phosphate, the mutant K72A of the 8-kDa domain and the full-lenghth protein beta-pol exhibit 90 and 80% loss of activity, respectively 692941 4.2.99.B1 K72A less than 10% of wild-type enzyme activity, Lys72 and Lys35 are involved in the dRP lyase reaction catalyzed by the 8-kDa domain and Lys35 is involved in 5'-phosphate recognition. Lys35 may stabilize the leaving group in the dRP lyase reaction through its interaction with the 5'-phosphate of the DNA-terminus 692940 4.2.99.B1 K72A mutant is not able to exhibit short-patch repair mechanism 692064 4.2.99.B1 K72A part of the active site, above 95% loss of enzyme activity 692010 4.2.99.B1 K72A PCR-based site-directed mutagenesis, deficient in forming a Schiff base intermediate and accumulating an enzyme dRP intermediate without releasing free dRP 692962 4.2.99.B1 K72R part of the active site, above 95% loss of enzyme activity 692010 4.2.99.B1 K84A retains wild-type enzyme activity 692940 4.2.99.B1 additional information an active-site deletion mutant of beta-pol, in which residues within the C-terminal portion, residues 263-335 that are required for DNA synthesis are deleted, completely deficient in single-nucleotide base excision repair DNA synthesis, but active in 5'-deoxyribose phosphate removal 694332 4.2.99.B1 additional information construction of PolB-knockout mouse embryonic fibroblasts (MEFs)Mbeta19tsA, phenotype. Mouse embryonic fibroblasts (MEFs) deficient in enzyme PolB show significantly increased sensitivity to methyl methanesulfonate (MMS). Wild-type MEFs and PolB-knockout MEFs are transfected with shRNA-expression vectors for Apex knockdown. Apex knockdown has essentially no effect on cell viability in the presence or absence of PolB. Thus, Apex knockdown does not affect the number of viable cells immediately after MMS treatment. The MMS sensitivity depends on Apex protein levels and suggest that resistance at low concentrations of MMS is due to an Apex-dependent/PolB-independent repair mechanism 748493 4.2.99.B1 additional information construction of several AtPolIB and deletion mutants, AtPolIB exo-DELTAins1, AtPolIB exo-DELTAins2, and AtPolIB exo-DELTAins3, AtPolIB exo- and polymerase mutants harboring deletions in insertion 1, 2 or 3, the mutants, especially AtPolIB exo-DELTAins1 and 3 insertion mutants, shows reduced activity compared to wild-type. Deletions in AtPolIB decrease strand-displacement activities 747641 4.2.99.B1 additional information the lyase catalytic pocket, Lys72, is replaced with each of several nonproteinogenic lysine analogues. The modified Pol beta enzymes are produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNACUA transcript, suppression of the UAG codon in the transcribed mRNA leads to elaboration of full-length Pol beta having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine affects mainly the stability of the Schiff base intermediate and results in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine at position 72, apparently shifts the amino group to a position less favorable for Schiff base formation. This effect is attenuated when the side chain is elongated by replacing one side-chain methylene group with a bridging S atom (thialysine). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an epsilon-amino group (homoarginine) or a sterically constrained secondary amine (piperidinylalanine) leads to almost complete suppression of dRP excision activity and the ability of Pol beta to support BER 747129 4.2.99.B1 R283A site-directed mutagenesis, C-terminal polymerase domain, deficient in base excision repair DNA synthesis activity of the polymerase, but has full dRP lyase activity 694332 4.2.99.B1 Y39F part of the active site, similar to wild-type enzyme activity 692010