Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis.
The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G>T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.
D'Agostino
Vito G
VG
Department of Genetics and Microbiology, University of Pavia, Pavia, Italy.
Minoprio
Anna
A
Torreri
Paola
P
Marinoni
Ilaria
I
Bossa
Cecilia
C
Petrucci
Tamara C
TC
Albertini
Alessandra M
AM
Ranzani
Guglielmina N
GN
Bignami
Margherita
M
Mazzei
Filomena
F
D011125:Adenomatous Polyposis Coli / Q000235:genetics*; D001483:Base Sequence; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
D058619:Maltose-Binding Proteins; D050505:Mutant Proteins; D033902:Periplasmic Binding Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase