Determination of regions within hNEIL1 involved in binding to the 9-1-1 complex

<p><b>Copyright information:</b></p><p>Taken from "The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates NEIL1 glycosylase"</p><p></p><p>Nucleic Acids Research 2007;35(8):2463-2472.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885643.</p><p>© 2007 The Author(s)</p> () Binding of hNEIL1 deletion mutants to GST-hRad9, GST-hRad1 and GST-Hus1. Immobilized GST-hHus1 (lane 2), GST-hRad1 (lane 3), GST-hRad9 (lane 4), and GST alone (lane 5) were incubated with a mixture of 100 ng each of purified processed intact hNEIL1 (residues 2–390), hNEIL1-CΔ40 (residues 2–350) and hNEIL1-CΔ101 (residues 2–289). The pellets were fractionated on a 10% SDS–PAGE followed by western blot analysis with the hNEIL1 antibody. Lane 1 contains 30 ng each of hNEIL1, hNEIL1-CΔ40, and hNEIL1-CΔ101 (30% of the total input). () Binding of hNEIL1 deletion mutants to His-tagged hHus1. His-hHus1 bound to His-Select (Sigma) magnetic beads (lanes 1–3) or Beads alone (lanes 4–6) were incubated with wild-type or mutant hNEIL1. The presence of hNEIL1 in the pellet was examined by western analysis with anti-NEIL1 antibody. () Far-western analysis. Processed intact and mutant hNEIL1 were separated by 10% SDS–PAGE and transferred onto nitrocellulose membrane. Lane 4 contains BSA. The proteins on the membrane were renatured and incubated with 10 pmol/ml of His-tagged hHus1. Subsequent western blotting was performed using His-tag antibody. () Graphic depiction of hNEIL1 constructs and the summary of binding results of these constructs with the 9-1-1 complex from A–C. The intact hNEIL1 contains 390 amino acid residues, however, processed intact hNEIL1 contains residues 2–390. hNEIL1-CΔ40 and hNEIL1-CΔ101 contain residues 2–350 and 2–289, respectively. The ‘+’ and ‘−’ listed on the right of each construct indicate positive and negative interactions with the 9-1-1 complex, respectively

Quantitative analyses of fold stimulation of hHus1, hRad1, hRad9 and the 9-1-1 complex expressed in the baculovirus system on processed full-length hNEIL1 (diamonds), NEIL1-CΔ40 (squares), and NEIL1-CΔ101 (triangles) activities from three experiments

<p><b>Copyright information:</b></p><p>Taken from "The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates NEIL1 glycosylase"</p><p></p><p>Nucleic Acids Research 2007;35(8):2463-2472.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885643.</p><p>© 2007 The Author(s)</p> The representative gels are shown in . The error bars reported are the standard deviations of the averages

HNEIL1 activity was stimulated by hHus1, hRad1, hRad9 and the 9-1-1 complex expressed in the baculovirus-transfected insect cells

<p><b>Copyright information:</b></p><p>Taken from "The human checkpoint sensor Rad9–Rad1–Hus1 interacts with and stimulates NEIL1 glycosylase"</p><p></p><p>Nucleic Acids Research 2007;35(8):2463-2472.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885643.</p><p>© 2007 The Author(s)</p> Human Hus1 (), hRad1 (), hRad9 () and the 9-1-1 complex () enhance the activities of processed full-length hNEIL1 and NEIL1-CΔ40, but not NEIL1-CΔ101. ()–(), Lane 1, thymine glycol (Tg)/A-containing DNA substrate and lane 2, 1.8 fmol (90 pM) of DNA substrate was incubated with hNEIL1 (1 nM). ()–(), Lanes 3–7 are similar to lane 2 but with added 3.125, 6.25, 12.5, 25 and 50 nM hHus1, hRad1, or hRad9, respectively. (), Lanes 3–7 are similar to lane 2 but with added 4, 8, 16, 32 and 64 nM of the 9-1-1 complex, respectively. Lanes 8–13 are similar to lanes 2–7 except using 1 nM hNEIL1-CΔ40. Lanes 14–19 are similar to lanes 2–7 except using 1 nM hNEIL1-CΔ101. The products were separated on a 14% DNA sequencing gel. Arrows mark the intact DNA substrate (I) and the βδ-elimination product (δ)

Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis-0

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis"</p><p>Nucleic Acids Research 2005;33(2):597-604.</p><p>Published online 26 Jan 2005</p><p>PMCID:PMC548354.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p

Functional interaction between hMutSα and wild-type and mutant hMYH proteins

<p><b>Copyright information:</b></p><p>Taken from "Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis"</p><p>Nucleic Acids Research 2005;33(2):597-604.</p><p>Published online 26 Jan 2005</p><p>PMCID:PMC548354.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The glycosylase assay was performed as in , except that the reactions contained different amounts of hMYH proteins and 0.25 or 2 nM of hMutSα. Lane 1–3, reactions contained 10 nM of wild-type hMYH with 0, 0.25 and 2 nM of hMutSα, respectively; lanes 4–6, reactions contained 109 nM of hMYH(R227W); and lanes 7–9, reactions contained 40 nM of hMYH(V232F). The arrows indicate the intact DNA substrate and nicked product

Additinal file 1:

In the supplement Material Section results from the protein purification and respective SDS-PAGE as well as the data from the stimulation of a SIRT6 mutant on MYH and APE1 activities are presented