Farida S. Sharief

Human DNA polymerase θ possesses 5′-dRP lyase activity and functions in single-nucleotide base excision repair in vitro

DNA polymerase θ (Pol θ) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol θ has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol θ has intrinsic 5′-deoxyribose phosphate (5′-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol θ is a ∼300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol θ possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5′-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5′-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH4 cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol θ. These results are consistent with a role of Pol θ in BER.

Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg2+ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.

Mapping of human lactate dehydrogenase-A, -B, and -C genes and their related sequences: the gene for LDHC is located with that for LDHA on chromosome 11

Human testis-specific lactate dehydrogenase-C (LDHC) gene-related sequences are located with the LDHA gene on chromosome 11. The LDHB gene is on chromosome 12. Chromosomes 1, 2, 4, 9, and 10 appear to contain LDHA gene-related sequences, whereas the X chromosome and chromosome 13 possess LDHB gene-related sequences.

Cancer-Associated Lactate Dehydrogenase is a Tyrosylphosphorylated Form of Human LDH-A, Skeletal Muscle Isoenzyme

Cancer-associated lactate dehydrogenase is a tyrosylphosphorylated form of human skeletal muscle isoenzyme, since the partial amino acid sequences of human liver LDH-K/A protein were found to be identical with the known primary structure of human LDH-A isoenzyme and the LDH-A isoenzymes from human placenta and bovine muscle were shown to be tyrosylphosphorylated. This tyrosylphosphorylated LDH-K/A protein was also found to be complexed with 21 kD, 30 kD, and 56 kD proteins.

cDNA cloning and genomic organization of enhancer of split groucho gene from nematode Caenorhabditis elegans.

This first genomic Enhancer of split groucho (ESG) gene and its full length complementary DNA (cDNA) from nematode C. elegans were cloned and sequenced via homology with the corresponding Drosophila groucho cDNA. The cDNA of 2.1‐Kb encodes a protein of 612 amino acids, and the nematode ESG protein is the smallest and most different in structure compared to all ESG related proteins. The gene isolated is 4,246‐bp in size, including 1,219‐bp promoter region. A putative TATA‐box at position ‐1166, two consensus sequence of ACTGG, characteristic of leader binding protein‐1 (LBP‐1) binding motifs at position ‐563 and ‐211 and nine CAAT boxes were found in the promoter region of ESG gene. The protein‐coding sequence is interrupted by five introns. The length of introns 1 to 5 is 52, 252, 87, 53 and 518 bp, respectively. The overall structural relationships of the ESG‐related proteins among human, mouse, rat, Xenopus, Drosophila and nematode were also analyzed.