Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V

Abstract

Oxanine (O) is a deamination product derived from guanine with the nitrogen at the N1 position substituted by oxygen. Cytosine, thymine, adenine, guanine as well as oxanine itself can be incorporated by Klenow Fragment to pair with oxanine in a DNA template with similar efficiency, indicating that oxanine in DNA may cause various mutations. As a nucleotide, deoxyoxanosine may substitute for deoxyguanosine to complete a primer extension reaction. Endonuclease V, an enzyme known for its enzymatic activity on uridine-, inosine- and xanthosine-containing DNA, can cleave oxanosine-containing DNA at the second phosphodiester bond 3' to the lesion. Mg2+ or Mn2+, and to a small extent Co2+ or Ni2+, support the oxanosine-containing DNA cleavage activity. All four oxanosine-containing base pairs (A/O, T/O, C/O and G/O) were cleaved with similar efficiency. The cleavage of double-stranded oxanosine-containing DNA was approximately 6-fold less efficient than that of double-stranded inosine-containing DNA. Single-stranded oxanosine-containing DNA was cleaved with a lower efficiency as compared with double-stranded oxanosine-containing DNA. A metal ion enhances the binding of endonuclease V to double-stranded and single-stranded oxanosine-containing DNA 6- and 4-fold, respectively. Hypothetic models of oxanine-containing base pairs and deaminated base recognition mechanism are presented.

Figure 1

Mutagenicity of oxanine in DNA. (A) Addition of dNTP to O-containing DNA by E.coli polymerase I Klenow Fragment (3′ exo-). Addition of dOTP and individual dNTP by Klenow Fragment was assayed as detailed in Materials and Methods. HEX, fluorophore (Integrated DNA Technologies) and Cntl, substrate control. (B) Extension of primer on O-containing template. The primer extension reactions were performed as detailed in Materials and Methods with indicated species of dNTPs.

Figure 2

Cleavage activity of purified Tma endo V on O-containing DNA. The cleavage reactions were performed as detailed in Materials and Methods with 1, 10 and 100 nM Tma endo V protein. Cleavage products were separated from remaining substrate by electrophoresis. M, 36mer marker; N = A, T, G or C; FAM, fluorophore (Integrated DNA Technologies); and -, control reactions without addition of endo V.

Figure 3

Effect of divalent metals on Tma endo V cleavage activity. The cleavage reactions containing 100 nM Tma endo V and various metal ions were incubated at 65°C for 30 min before electrophoresis. (A) Electrophoresis diagram of metal ion dependency. (B) Quantitative analysis of cleavage activity with various metal ions. (C) Titration of Mg²⁺ with wt Tma endo V and T/O substrate. (D) Titration of Mn²⁺ with wt Tma endo V and T/O substrate. (E) Titration of Mn²⁺ with wt Tma endo V and T/I substrate. (F) Titration of Mn²⁺ with D43A mutant Tma endo V and T/O substrate.

Figure 4

Time course analysis of T/O and T/I cleavage by Tma endo V. Cleavage reactions were performed as described in Materials and Methods with 1 nM Tma endo V (triangles), 10 nM (circles) and 100 nM (squares). Reactions were stopped on ice at indicated time points and followed by adding equal volume GeneScan stop buffer. (A) Representative GeneScan gel analysis of T/O cleavage (E:S = 10:1). (B) Plots of T/O cleavage. (C) Representative GeneScan gel analysis of T/I cleavage (E:S = 1:1). (D) Plot of T/I cleavage (E:S = 1:1).

Figure 5

Gel mobility shift analysis of Salmonella endo V with double-stranded O-containing substrate. Lanes: 1, 0 nM endo V; 2, 10 nM endo V; 3, 50 nM endo V; 4, 100 nM endo V; 5, 250 nM endo V; and 6, 500 nM endo V. (A) Binding of T/O substrate without metal. (B) Binding of T/O substrate with Ca²⁺. (C) Quantitative analysis of endo V binding to T/O substrate without metal (squares) or with Ca²⁺ (circles).

Figure 6

Time course analysis of single-stranded O and I cleavage by Tma endo V. Cleavage reactions were performed as described in Materials and Methods with 1 nM Tma Endo V (triangles), 10 nM (circles) and 100 nM (squares). Reactions were stopped on ice at indicated time points and followed by adding equal volume of GeneScan stop buffer. (A) Representative GeneScan gel analysis of single-stranded O cleavage (E:S = 10:1). (B) Plots of single-stranded O cleavage. (C) Representative GeneScan gel analysis of single-stranded I cleavage (E:S = 1:1). (D) Plot of single-stranded I cleavage (E:S = 1:1).

Figure 7

Gel mobility shift analysis of Salmonella endo V with single-stranded O-containing substrate. Lanes: 1, 0 nM endo V; 2, 10 nM endo V; 3, 50 nM endo V; 4, 100 nM endo V; 5, 250 nM endo V; and 6, 500 nM endo V. (A) Binding of single-stranded O-containing substrate without metal. (B) Binding of single-stranded O-containing substrate with Ca²⁺. (C) Quantitative analysis of endo V binding to single-stranded O-containing substrate without metal (squares) or with Ca²⁺ (circles).

Figure 8

Models of oxanine base pairs and deamianted base recognition mechanism. (A) Proposed O-containing base pairs. (B) A hypothetic model of deamianted base recognition mechanism.