T 4 噬菌体 DNA 的复制与调控

1. T4噬菌体DNA的复制与调控 吴俊 张年辉 卿人韦

2. T4噬菌体DNA的复制与调控 origin-dependent replication early in infection recombination-dependent replication at later times (RDR) • replication mediator protein (RMP)----gp59 and uvsY • the sliding clamp-----gp45

3. Fig.2-1 Model of a replication fork with bacteriophage T4 proteins

4. Fig.1- 1 T4 in vitro system for RDR

5. Features of the RDR pathway First is the strict requirement, under physiological conditions, for an RMP protein(uvsY) to promote the uvsX-catalyzed initiation of leading strand synthesis via the D loop-forming mechanism mentioned above. Second is the requirement for another mediator protein(gp59) to initiate the lagging strand synthesis component of RDR. Third is the intriguing observation, diagrammed in Fig. 1-1, that the synthesis of Okazaki fragments always occurs on the displaced strand of the D loop, and not on the 59 extension of the invading ssDNA.

6. Role of UvsY Protein in Assembly of the T4 Presynaptic Filament UvsY helps uvsX displace gp32 from ssDNA, a reaction necessary for proper formation of the presynaptic filament. UvsY interacts with and stabilizes uvsX-ssDNA filaments after they are assembled. Note: uvsX recombinase cooperatively bound to ssDNA

7. Fig.1-2A Biochemical model for uvsY-mediated assembly of the T4 presynaptic filament

8. Hypothesis of Presynapsis Coupled to the Nucleolytic Resection of dsDNA Ends 3’ ssDNA tails generated during T4 origin-dependent replication are natural primers for RDR because the presence of homology is guaranteed by the terminal redundancy of T4 DNA .Several other mechanisms exist for 3’ tail generation, including nucleolytic resection of DNA double-stand breaks (DSBs). Both DSB repair and ‘‘normal’’ RDR processes depend on the T4 gp46 and gp47 proteins. The observation of a strong protein–protein interaction between gp46/47 and uvsY raises another intriguing possibility: that nucleolytic resection of DSBs is directly coupled to the assembly of a presynaptic filament on the remaining strand. A hypothetical model for this process is shown in Fig. 1-2B.

9. Fig.1-2B Hypothetical model for presynapsis coupled togp46y47-catalyzed resection of a DSB

10. Structure of T4 Gene 59 Helicase-Loading Protein (gp59) T4 59 helicase-loading protein is a small, basic, almost completely α-helical protein whose N-terminal domain has structural similarity to high mobility group family proteins(HMG). Its 13 α-helices are divided into N and C domains of similar size. The single short β-sheet connects N-terminal residues 2–4 with residues 197–199 near the C terminus. There is a narrow groove between the two domains on the ‘‘top’’ of the protein. The surface of the protein is notable for the high density of basic and hydrophobic residues, which may be important for its DNA and protein interactions (Fig. 2-2). Gp59 recognizes specific structures rather than specific sequences. It binds and loads the helicase on replication forks and on three- and four-stranded (Holliday junction) recombination structures,without sequence specificity.

11. Fig. 2-2Ribbon diagrams of the crystal structure of the T4 gene 59 helicase-loading protein showing its structural similarity with HMG proteins.

12. Fig.1-3 Biochemical model for gp59-ediated helicase assembly at T4 replication fork

13. Fig. 1-4 Enzyme partitioning model for strand-specific priming of Okazaki fragments during T4 recombination-dependent replication

14. DNA polymerase holoenzyme The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep. It proceeds through 10 steps and 7 conformational changes in gp45.

15. Scheme 1 Steps in the Formation of the T4 Holoenzyme.

16. Fig.3- 1(A) X-ray crystal structure of gp45 showing the interdomain connecting loop and the subunit interface. (B) In-plane model of opening of gp45 showing the location of the mutations: V163C in blue, S158C in green, and T168C in pink, with the donor tryptophan in orange. Each mutation is shown only once for clarity. (C) Out-of-plane model of opening.

17. Fluorescence Resonance Energy Transfer 运用Fluorescence resonance energy transfer(FRET)技术观测了gp45在T4DNA polymerase holoenzyme组装过程中的动态变化，并设计了gp45的动态图。该方法的原理是：gp45的一个亚基上有一个内源色氨酸（W91)，它在能量转移中能够被检测，将它作为荧光团供体。还设计了gp45特异位置上的三个突变体（V163C,S158C,T168C),作为荧光团受体。通过计算供体和受体间的距离，得出了gp45在全酶形成中的开启和关闭模型 (Fig.3-5 )。

18. Fig.3-5 Molecular models of gp45

19. 谢谢