PAGE 5 OF 6 Nucleic Acids Research, 2007, Vol. 35, No. 15 e100 Figure 5. Electrophoretical analysis of the products. Upper (left) or lower (right) strand of 17-bp substrate dsDNA was 50-radio labeled and reacted with indicated concentrations of CTD and subjected to electrophoresis on an 11% polyacrylamide sequencing gel. Lane 1: pyrimidine-speciﬁc sequence marker generated by Maxam–Gilbert method; lane 2: purine-speciﬁc marker; lane 3–6: upper strand labeled substrates were digested with 0, 50, 100 and 200 nM CTD, respectively; lane 7–10: lower strand labeled substrates were digested with 0, 50, 100 and 200 nM CTD, respectively. Numbers to the left and right of the gel indicate the length (bases) of the DNA seize markers and products, respectively. and the nature of cleaved sites would be a great help in understanding the mechanism of an enzyme and its role in a biological process. For example, when a newly discovered nuclease preferably incised at an abasic site yielding 50-deoxyribosephosphate (50-dRP) end, we should consider the possibility that the nuclease takes part in base excision repair through the repair of abasic sites and the repair pathway requires an enzyme that have a 50-dRPase activity such as DNA polymerase b. ACKNOWLEDGEMENTS This work was supported in part by Grants-in-aid for Scientiﬁc Research to R.M. from the Ministry of Education, Science, Sports and Culture of Japan and by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan. Funding to pay the Open Access publication charges for this article was provided by the RIKEN Structural Genomics/Proteomics Initiative. Figure 6. CTD did not demonstrate a 30–50 exonuclease activity. The substrate dsDNA whose 30-terminus was blocked or unblocked with LNA was incubated with CTD (A) or KOD polymerase (B) and subjected to electrophoresis on an 11% polyacrylamide sequencing gel. Lanes 1–4: unblocked substrates were digested with 0, 50, 100 or 200 nM CTD, respectively; lanes 5–8: blocked substrates were digested with 0, 50, 100 or 200 nM CTD, respectively; lanes 9 and 10: unblocked substrates were digested with 0 or 5 nM KOD polymerase; lanes 11 and 12: blocked substrates were digested with 0 or 5 nM KOD polymerase. The length of the product fragments (bases) are indicated by numbers to the right of panel A. Conﬂict of interest statement. None declared. REFERENCES 1. Lieberk,M.R. (1997) The FEN-1 family of structure-speciﬁc nucleases in eukaryotic DNA replication, recombination and repair. Bioessays, 19, 233–240. 2. West,S.C. (2003) Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell. Biol., 4, 435–445. 3. Grindley,N.F., Whiteson,K.L. and Rice,P.A. (2006) Mechanism of site-speciﬁc recombination. Annu. Rev. Biochem., 75, 567–605. 4. Marti,T.M. and Fleck,O. (2004) DNA repair nucleases. Cell. Mol. Life. Sci., 61, 336–354. 5. Andersen,J.S., Svensson,B. and Roepstorﬀ,P. (1996) Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: powerful analytical tools in recombinant protein chemistry. Nat. Biotechnol., 14, 449–457. 6. Little,D.P., Thannhauser,T.W. and McLaﬀerty,F.W. (1995) Veriﬁcation of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry. Proc. Natl Acad. Sci. USA, 92, 2318–2322. 7. Oberacher,H., Niederstatter,H. and Parson,W. (2006) Liquid chromatography-electrospray ionization mass spectrometry for simultaneous detection of mtDNA length and nucleotide poly- morphisms. Int. J. Leg. Med., 121, 57–67. 8. Null,A.P. and Muddiman,D.C. (2001) Perspectives on the use of electrospray ionization fourier transform ion cyclotron resonance mass spectrometry for short tandem repeat genotyping in the post- genome era. J. Mass Spectrom., 36, 589–606. 9. Eisen,J.A. (1998) A phylogenomic study of the MutS family of proteins. Nucleic Acids Res., 26, 4291–4300. 10. Modrich,P. (1989) Methyl-directed DNA mismatch correction. J. Biol. Chem., 264, 6597–6600.