Surface plasmon resonance spectroscopy and quartz crystal microbalance study of MutS binding with single thymine-guanine mismatched DNA.
MutS is a DNA mismatch binding protein that recognizes heteroduplex DNA containing mispaired or unpaired bases. In this study, we employ a quartz crystal microbalance (QCM) and a surface plasmon resonance (SPR) device for the study of MutS binding with DNA containing a single Thymine-Guanine (T-G) mismatch at different sites. Multi-step surface binding reactions are involved in the study, including probe DNA immobilization on the sensor surface through biotin-streptavidin-biotin bridge chemistry, target DNA hybridization to form T-G heteroduplexes, and MutS recognition of the mutation sites. The QCM frequency (d f) and motional resistance (d R, an impedance parameter reflective of QCM damping), as well as the SPR angle shift (d q ) are recorded for the binding reactions. The combined SPR and QCM data collection and analysis allow for an assessment of not only the amount of bound biopolymer but provide also information on also the structural properties of the streptavidin, DNA and MutS/DNA complexes. The affinity of the MutS/T-G heteroduplex assembly is determined by both the QCM and SPR methods through titration of the surface bound DNA with increasing MutS concentration. It is found that if the T-G mismatch is in the center of the DNA fragment, the MutS/DNA complex is more stable than if the mismatch is located near the end of the fragment.
Su
Xiaodi
X
Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602. xd-su@imre.a-star.edu.sg <xd-su@imre.a-star.edu.sg>
Wu
Ying-Ju
YJ
Robelek
Rudolf
R
Knoll
Wolfgang
W
D020137:Base Pair Mismatch; D003460:Crystallization; D004247:DNA / Q000032:analysis*; D006147:Guanine / Q000737:chemistry*; D007700:Kinetics; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry*; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000379:methods*; D013824:Thermus / Q000378:metabolism; D013941:Thymine / Q000737:chemistry*
D009692:Nucleic Acid Heteroduplexes; D011108:Polymers; D006147:Guanine; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein; D013941:Thymine