Kinetic mechanisms of rat polymerase β-ssDNA interactions. quantitative fluorescence stopped-flow analysis of the formation of the (pol β)16 and (pol β)5 ssDNA binding mode

Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex

“…[18][19][20][21][22][23][24][25][29][30][31]58,59 The data reported here show that, in spite of very different structures, both enzymes show significant similarity in their interactions with the ssDNA and dsDNA. The total DNA-binding site is structurally and functionally heterogeneous.…”

“…Moreover, the high dsDNAaffinity of the subsite, which is engaging in interactions with the dsDNA, indicates that this is the proper DNA-binding subsite. 23,24,[29][30][31]58,59 ASFV pol X binds dsDNA exclusively using its non-catalytic C-terminal domain: the location of the proper DNA-binding subsite of the enzyme As pointed out above, the NMR structure alone does not provide any indication as to which of the lysine-rich regions serves as the proper DNAbinding site, i.e. it is used exclusively by ASFV pol X in binding to the dsDNA.…”

“…17 Similar heterogeneity of the total DNA-binding site has been found for the mammalian pol β. 23,24,[29][30][31]58,59 As a result, pol β forms different binding modes with the ssDNA, where the enzyme occludes different numbers of nucleotides by engaging one or both DNA-binding subsites. However, ASFV pol X does not form different binding modes with ssDNA, an indication that the DNA-binding subsites of the enzyme are much less autonomous than the analogous subsites of pol β.…”

“…Such correspondence of activities between two structurally very different enzymes points to a general functional behavior of the DNA repair polymerases, where the total DNA-binding site is composed of two DNA-binding subsites, which play very different functions in ssDNA gap recognition and catalysis. [18][19][20][21][22][23][24][25][29][30][31]58,59 Materials and Methods…”

DNA Polymerase X From African Swine Fever Virus: Quantitative Analysis of the Enzyme–ssDNA Interactions and the Functional Structure of the Complex

“…19,21,[28][29][30][31] The autonomous nature of the DNA-binding subsite on the 8 kDa domain allows pol b to bind small areas of the DNA substrate without engaging the catalytic 31 kDa domain of the enzyme into interactions with the nucleic acid. [15][16][17][18][19][20][21][28][29][30][31] The subsequent association of the 31 kDa domain with the available DNA, follows this initial binding process. [28][29][30][31] However, ASFV pol X does not possess the 8 kDa domain or an equivalent structural element, which would suggest a mechanism of binding similar to pol b (Figure 1).…”