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My research is about DNA metabolism and several interesting projects in other areas. Since most of my projects about DNA metabolism are not ready for publication yet, I can only briefly introduce my major project and two interesting little projects. Eukaryotes have chromatinized genome with the nucleosome as the basic building block. In eukaryotes, DNA metabolism including DNA repair takes place in chromatin rather than in free DNA duplex. So, it is critical to understand DNA repair in chromatin. As the carrier of genetic information, DNA is quite vulnerable. Many DNA repair systems are required to maintain genome stability. Including base excision repair, mismatch repair system, and many other systems. My project is focusing on base excision repair in chromatin. The base excision repair pathway handles base damages caused by alkylation, oxidation, and deamination of nucleobase. DNA glycosylase can locate and remove damaged nucleobases in DNA, leaving an AP site. Which will be cleaved by AP site endonuclease APE1. Leaving a nick on DNA which will be repaired by DNA polymerase and DNA ligase. The structures of these repair factors are solve to high resolution by X-ray crystallography. Since we already know how base excision repair factors act on free DNA from crystal structures. Now we turned our eyes to nucleosome. Nucleosome is known for restricting DNA accessibility, but many studies have shown base excision repair is active on nucleosome. So the key question in chromatin-based base excision repair is how repair factors read the chromatinized genome. Here shows some structural studies trying to answer how protein factors read the chromatinized genome. Such as the retroviral intasome of retroviral DNA integration in nucleosome, UV-DDB complex of nucleotide excision repair initiation in nucleosome, and transcription factors OCT4-SOX2 binding to nucleosome. They found that protein factors can introduce local DNA distortion and register shift to nucleosomal DNA to access specific DNA site. To answer our own question, I collaborated with an EM expert in our lab, we solve several cryo-EM structures of DNA base damage-bearing nucleosomes in complex with DNA glycosylase A A G. Which represents the first step of base excision repair. Another group in the U.S. solved the structure of APE1 in complex with nucleosome. Which represents the second step of base excision repair. In this project, we used hypo xanthine as the substrate of A A G. We placed one hypo xanthine in four different positions to make four different nucleosomes. Two of these are solvent-facing positions with high solution accessibility, which are -30 and -50. One is an occluded position with medium solution accessibility, which is -53. The last one is an embedded position with low solution accessibility, which is -55. Previous in vitro studies showed that A A G is active in all four positions, and we want to know how A A G does that. We purified recombinant human A A G from E.coli. And we assembled four single-hypo xanthine-containing nucleosomes. We combined them into stable complexes by GraFix. We collected four sets of data, and solved two structures from each dataset. Here shows the -30 dataset, we got a 2.8 A Apo nucleosome, and a 2.9 A A A G-NCP complex. Here shows the overall structure of A A G-NCP -30 complex. As you can see, A A G binds to nucleosomal DNA without interacting with the octameric core. A protruding β hairpin is inserted into the minor groove of DNA for damage recognition. We built the atomic model based on our cryo-EM map. And found extensive interactions between A A G and nucleosome covering 8 base pairs. An interesting fact is that we placed one hypo xanthine in our nucleosome, but our cryo-EM map indicates an AP site in -30. And the presence of hypo xanthine causes steric clashes. Suggesting our structure is in a post-catalytic state. The post-catalytic AP site is flipped-out, which is usually called base-flipping. A A G captures the flipped AP site through multiple interactions. The difference between our apo nucleosome and canonical nucleosome is that our structure contains one hypo xanthine. We compared these two structures and found apparent DNA deformation around -30. We quantified the DNA deformation by calculating the RMSD value between damaged-strand DNA backbone and found that the presence of one hypo xanthine leads to global perturbation of nucleosomal DNA. The average RMSD is around 1 A. The smallest deformation is found around dyad axis of nucleosome and displacement increases toward DNA end. We also calculated the RMSD between A A G-NCP-30 complex and apo nucleosome. Instead of global perturbation, we observed apparent local DNA distortion after A A G binding. The maximum RMSD is exactly at -30 with a value of 4.4 A. The local DNA distortion based binding mechanism is also utilized by OCT4-SOX2 and retroviral intasome while accessing nucleosomal DNA. We want to know whether A A G accesses nucleosomal DNA by imposing local DNA distortion in all positions. And we analyzed other three datasets. For the -50 datasets. We got a 2.9 A apo nucleosome and a 3.0 A A A G-NCP-50 complex. We found similar global perturbation of nucleosomal DNA in the apo nucleosome with an average RMSD around 1 A. And the local DNA distortion in A A G-NCP-50 complex is also similar to A A G-NCP-30 complex. The maximum RMSD is about 3 A at -50. So we think at least for solvent facing positions, A A G utilizes local DNA distortion to access damaged bases. But for -53 and -55 which are not solvent-facing positions, the situation might be different. For the -53 dataset, -53 is an occluded position, we got a 2.8 A apo nucleosome and a 3.1 A A A G-NCP-53 complex. We observed almost identical global DNA perturbation in the apo nucleosome. But for the A A G-NCP-53 complex, things are quite interesting. We found apparent local DNA distortion and local DNA register shift around -53. The -53 is now at -52 after A A G binding. The local register shift is quite easy to understand. The damaged base will be flipped out by A A G for A A G catalysis. With the original DNA register, base-flipping at -53 will result in clashes with histone H2B. By moving to -52, the steric clashes can be avoided. The shift is local, as you can see here. For the embedded -55 position, we got a 2.8 A apo nucleosome and an unusual nucleosome structure at 2.9 A. The global perturbation in the apo nucleosome is also similar to the other three apo structures. The unusual nucleosome structure is what we found interesting. The hypo xanthine is at -55, but we couldn’t get the density of DNA from -50 to DNA end which is quite unusual. This indicates this portion of DNA is very flexible. We know that nucleosome structure is dynamic, partial opening of DNA end occurs frequently. But these dynamic structural intermediates are very difficult to capture using cryo-EM. We think it’s the A A G binding to damaged base at -55, making the partial opening process less reversible, allowing us to capture the structure. After analyzing the four sets of structures, we found that A A G utilizes different mechanisms to access damaged base in nucleosome. But the global perturbation of nucleosomal DNA in apo nucleosome structures are identical. We want to know what that means. We think it represents the destabilization of nucleosome due to the presence of hypo xanthine. We calculated the buried surface area between nucleosomal DNA histone octamer to quantify the interaction between DNA and histones. Compared to canonical NCP, the buried surface area in hypo xanthine-containing apo nucleosomes decreased almost 15%, which indicates weaker interaction between DNA and histones. Suggesting the destabilization of nucleosome. Based on our results. We proposed a working model for A A G on nucleosome. The presence of damaged base at any position in nucleosome will perturb nucleosomal DNA and destabilize nucleosome. For solvent-facing positions, A A G utilizes local DNA distortion to access damaged base. For occluded positions, additional local register shift of DNA required. While for embedded positions, A A G utilizes the dynamic nature of nucleosome and captures damaged base in partially opened DNA end.