哈佛大学分子生物学--DNA复制(第二讲).ppt

1、免疫学信息网,免疫学信息网,Looping the lagging strand to make both polymerases move in the same direction,免疫学信息网,The discovery of DNA polymerase. Arthur Kornberg and Bob Lehman pursued an enzyme in bacterial extracts that would elongate a chain of deoxyribonucleic acid just like glycogen synthase elongates a cha

2、in of glycogen. The enzymatic activity was unusual: 1) Needed a template which dictates what nucleotide was added: substrate was directing enzymatic activity 2) Needed a primer annealed to the template.,免疫学信息网,免疫学信息网,免疫学信息网,免疫学信息网,Wait a minute! John Cairns mutated the gene for DNA polymerase, polA,

3、 and the bacteria grew just fine! Either the polymerase hypothesis was all wrong, or there were other DNA polymerases in E. coli that carried out DNA synthesis in the polA strains.,免疫学信息网,免疫学信息网,免疫学信息网,Which polymerase is processive?,免疫学信息网,POLIII, b subunit,PCNA,免疫学信息网,Clamp loaders hydrolyze ATP t

4、o load clamp,How does one prove that the clamp ring is opened during loading?,免疫学信息网,免疫学信息网,免疫学信息网,Structure of a DNA polymerase (gp43 from phage RB69),Side view: Polymerase active site,Top view with template-primer: Polymerase site And proofreading site,免疫学信息网,* Topoisomerases II change the linking

5、 number in steps of 2 by passing both strands of double-stranded DNA through a break. * Eukaryotic topoisomerases isolated to date only relax supercoiled DNA, while prokaryotic topoisomerases (gyrases) can, given ATP, add supercoils. * TopoII releases catenated daughter molecules at the end of repli

6、cation. Inhibitors like etoposide are used in chemotherapy.,Topoisomerases relax DNA by changing the DNA linking number,免疫学信息网,* Topoisomerases I change the linking number in steps of 1. They pass a single DNA strand through a nick.Topoisomerase I is a protein of the metaphase chromosome scaffold. *

7、 In interphase, topoisomerase is bound to the nuclear matrix. * The DNA replication machinery also appears bound to the matrix. * Inhibitor (camptothecin) also used in chemotherapy.,免疫学信息网,Topoisomerase action can be divided into three steps: nicking (1), strand passage (2); resealing (3).,免疫学信息网,1,3,2,4,Cycle of topoisomerase activity infer