The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains

Abstract

Prior sequence analysis studies have suggested that bacterial ribonuclease (RNase) Ds comprise a complete domain that is found also in Homo sapiens polymyositis-scleroderma overlap syndrome 100 kDa autoantigen and Werner syndrome protein. This RNase D 3'-->5' exoribonuclease domain was predicted to have a structure and mechanism of action similar to the 3'-->5' exodeoxyibonuclease (proofreading) domain of DNA polymerases. Here, hidden Markov model (HMM) and phylogenetic studies have been used to identify and characterise other sequences that may possess this exonuclease domain. Results indicate that it is also present in the RNase T family; Borrelia burgdorferi P93 protein, an immunodominant antigen in Lyme disease; bacteriophage T4 dexA and Escherichia coli exonuclease I, processive 3'-->5' exodeoxyribonucleases that degrade single-stranded DNA; Bacillus subtilis dinG, a probable helicase involved in DNA repair and possibly replication, and peptide synthase 1; Saccharomyces cerevisiae Pab1p-dependent poly(A) nuclease PAN2 subunit, required for shortening mRNA poly(A) tails; Caenorhabditis elegans and Mus musculus CAF1, transcription factor CCR4-associated factor 1; Xenopus laevis XPMC2, prevention of mitotic catastrophe in fission yeast; Drosophila melanogaster egalitarian, oocyte specification and axis determination, and exuperantia, establishment of oocyte polarity; H.sapiens HEM45, expressed in tumour cell lines and uterus and regulated by oestrogen; and 31 open reading frames including one in Methanococcus jannaschii . Examination of a multiple sequence alignment and two three-dimensional structures of proofreading domains has allowed definition of the core sequence, structural and functional elements of this exonuclease domain.

Positive and Negative Regulation of Poly(A) Nuclease

“…By using two-hybrid analysis, we have demonstrated that Pab1p interacts with Pan3p and Pbp1p, two proteins that respectively appear to act as positive and negative regulators of PAN activity (9,23). Not surprisingly, our experiments have also shown that these two proteins also interact with the Pan2p subunit of PAN, i.e., the subunit whose homologies to previously characterized exonucleases (8,26) suggest that it harbors the catalytic activity relevant to PAN function. Figure 9 summarizes this set of interactions and raises interesting possibilities for the precise role of Pab1p in poly(A) trimming.…”

Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant

“…The T. gondii XPMC2 (TgXPMC2) cDNA encodes a protein of 361 amino acids with a predicted molecular weight of ª40 kDa and an isoelectric point of 9.59 (accession number AY841997). The N-terminal half of the protein encodes a putative exonuclease domain (residues 60-219) conserved in all XPMC2-related genes (Moser et al, 1997) (Fig. 3).…”

Mouse CAF1 Can Function As a Processive Deadenylase/3′–5′-Exonuclease in Vitro but in Yeast the Deadenylase Function of CAF1 Is Not Required for mRNA Poly(A) Removal

“…Higher eukaryotes also contain the PARN deadenylase, which is a principle component of cell-free extracts capable of efficient poly(A) shortening (15). Both PAN2 and PARN are members of the DEDD family of RNases (16,17). PAN2 is specifically activated by the poly(A)-binding protein (PAB1), whereas PARN becomes activated in the presence of the mRNA cap structure (15).…”