TY - JOUR
T1 - Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search
AU - Beckwitt, Emily C.
AU - Jang, Sunbok
AU - Carnaval Detweiler, Isadora
AU - Kuper, Jochen
AU - Sauer, Florian
AU - Simon, Nina
AU - Bretzler, Johanna
AU - Watkins, Simon C.
AU - Carell, Thomas
AU - Kisker, Caroline
AU - Van Houten, Bennett
N1 - Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2’-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.
AB - Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2’-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.
UR - http://www.scopus.com/inward/record.url?scp=85081600067&partnerID=8YFLogxK
U2 - 10.1038/s41467-020-15168-1
DO - 10.1038/s41467-020-15168-1
M3 - Article
C2 - 32170071
AN - SCOPUS:85081600067
SN - 2041-1723
VL - 11
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 1356
ER -