Human DNA Polymerase β Deoxyribose Phosphate Lyase

Abstract

DNA polymerase ␤ (␤-pol) cleaves the sugar-phosphate bond 3 to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5-incised by AP endonuclease, resulting in a 5 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the aminoterminal 8-kDa domain of ␤-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of ␤-pol are similar. However, k cat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The fulllength enzyme and the 8-kDa domain efficiently remove the 5 dRP from a preincised AP site in the absence of Mg 2؉ , and the pH profiles of ␤-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys 72 ) as the Schiff base nucleophile during lyase chemistry.The cellular genome suffers extensive damage from exposure to ultraviolet light and ionizing radiation and also from alkylating agents and reactive oxygen species that accumulate in cells due to environmental stress and natural metabolic processes (1). These DNA lesions occur at a frequency far too high to be compatible with life unless they are repaired to allow faithful function and reproduction of the genome. To remove such damage and restore the integrity of the genome, specific DNA repair pathways have evolved. The major pathway, which protects cells from the deleterious effects of nucleotide base DNA damage, is known as DNA base excision repair (BER). BER is initiated by removal of a damaged or inappropriate base residue in DNA by cleavage of the N-glycosidic bond (1, 2). If not repaired, the resulting abasic or apurinic/apyrimidinic (AP) site may result in mutations, altered gene expression, chromosome breakage, apoptosis, and/or cell death.Both procaryotes and eucaryotes possess class II AP endonucleases that cleave 5Ј to an AP site leaving a 3Ј-hydroxyl and a 5Ј 2-deoxyribose 5-phosphate (dRP) in the nicked DNA (Fig. 1). In Escherichia coli, two major AP endonuclease genes, xth and nfo, have been identified encoding exonuclease III and endonuclease IV, respectively (3, 4). Homologous genes to xth have been cloned from many sources, including Drosophila melanogaster and human cells (5-7). The major AP endonuclease in yeast, APN1, was shown to be an endonuclease IV homologue (8). These class II AP endonucleases also possess 3Ј-phosphodiesterase and 3Ј-phosphatase activities, and some members of the xth family carry 3Ј to 5Ј exonuclease activity (9). Subsequent to AP endonuclease cleavage in BER, the cleaved AP site must be processed further (Fig. 1). The enzymatic removal of the sugar-phosphate residue ...

Contributions of an Endonuclease IV Homologue to DNA Repair in the African Swine Fever Virus

“…We asked how the gap-filling and nick-editing activities are influenced when dRP, rather than phosphate, is the 5′ moiety of a gap or nick. Assays employing the THF analogue of 5′-dRP (5′-P i -THF) 5 are shown in Figure 10. Burst assays were employed with Pol X because this allowed an evaluation of the pre-steady state in addition to the steady state.…”

“…This apparent discrepancy might best be accounted for by nonspecific affects in the cell-free extract, such as inactivation of ASFV APE by a metalloprotease; an alternative explanation, that the A:THF substrate was rendered unusable by an E. coli protein, seems unlikely since the electrophoretic properties of the substrate were unperturbed. 5 Because this lacks the C1′ hydroxyl of dRP, it is not prone to spontaneous -elimination. For the reactions shown in A-C, 13 nM ASFV APE was incubated with 300 nM DNA substrate at 37°C for the indicated length of time; for the NIR assays in part D, protein concentrations were increased to 130 and 520 nM for ASFV APE and E. coli endonuclease IV, respectively.…”

Influence of DNA Structure on DNA Polymerase β Active Site Function

“…K d ) for the binding of heteropolymeric DNA gapped substrates were determined by inhibition of pol ␤ activity on a homopolymeric DNA as described previously (16). Enzyme activities were typically determined using a standard reaction mixture (50 l) containing 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 5 mM MnCl 2 , 30 M [␣-32 P]dTTP, 30 or 300 nM poly(dA)-poly(dT) 20 (K m ; expressed as 3Ј-OH primer termini), and varying concentrations of competitor heteropolymeric DNA.…”

The Domain Organization and Properties of Individual Domains of DNA Topoisomerase V, a Type 1B Topoisomerase with DNA Repair Activities

“…6B) activities. Similar to DNA polymerase ␤ (20,21,26), the lyase activity of Topo78 and Topo34 dRP is catalyzed via ␤-elimination as opposed to hydrolysis. We determined this activity by trapping the proteins with preincised AP DNA by using NaBH 4 as the Schiff base-reducing reagent.…”