1. Metabolism of DNA : Replication and Recombination

2. 6.1 DNA Replication
Metabolism of DNA : replication, repair, recombination and degradation.
Replication : semi-conservative replication, need nucleic acid precursors(NTPs), DNA polymerase.
Replication take place in two steps :
the DNA double helix is unwound(helicase),
new nucleotides are linked(DNA polimerase).

3. 6.1 DNA Replication
FIGURE 6.1 An overview of the components involved in replication of DNA in eukaryotes.Drawing by Mariana Ruiz Villarreal deposited at Wikipedia
In the cell, each separate DNA molecule has at least one site in the sequence that acts as the starting point

4. Topoisomerase : relieve helical tension
6.1.1 DNA Helicase
Helicase : break the force that hold the two DNA strands together(by ATP hydrolysis).
Single strand-binding protein assist the DNA helicase to stabilize the open structure.
Topoisomerase : relieve helical tension
Helicases are essential for replication, recombination, repair, transcription and translation.

5. Three conserved domains Helicase RecQ-C-terminal
6.1.1 DNA Helicase
Helicase can be grouped in different family based on conserved motif level
Members of the RecQ helicase family are found in all organism and mostly used in DNA repair.
Three conserved domains
Helicase
RecQ-C-terminal
Helicase-and-RNaseD-like-C-terminl

6. RecA-like modules are the cores of the helicase motor domain
6.1.1 DNA Helicase
RecA-like modules are the cores of the helicase motor domain
The RecQ-Ct and HRDC domains are substrate specificity, targeting and interaction with other protein.
FIGURE 6.2 Left: The structure of RecQ from E. coli. The RecA-like helicase domains are shown in green and yellow and the RecQ-Ct domain is shown in turquoise. Right: The ATP binding site and the residues involved in catalysis. Lys53 is from the P-loop and Asp146 and Glu147 are from the second conserved motif (DEXH). Arg329 from the other domain is close to the active site, similar to arginine fingers in other P-loop containing proteins (PDB: 1OYY).

7. 6.1.2 DNA polymerase
DNA polymerases are the key enzymes in assuring the accuracy in replication, repair and recombination.
Shape : roughly like an open right hand with a palm, a thumb and fingers.
DNA polymerase can be grouped into seven different classes : A, B, C, D, X, Y and RT(Table 6.2).

8. Active site located on palm sub-domain.
6.1.2 DNA polymerase
Active site located on palm sub-domain.
The thumb part bind DNA and fingers are important in the recognition and binding of nucleotides.
FIGURE 6.3 The structure of the T7 DNA polymerase. The structure can be described as a right hand. The palm is yellow, the fingers are blue and the thumb is red. The catalytically important magnesium ions are shown in yellow. The Exo domain (gray) is involved in proofreading. The DNA primer and template are colored orange and green, respectively.

9. All classes have similar basic structure(except D).
6.1.2 DNA polymerase
All classes have similar basic structure(except D).
Conserved structure in palm domain but finger and thumb structures are variable(classes A, B,Y and RT).
FIGURE 6.4 The structures of DNA polymerase families A, B, X and C. Top left: Pol I, Klenow
fragment (class A, PDB: 1KFS). Top right: DNA polymerase from phage RB69 (class B, PDB:
1IH7). Bottom left: Pol β (class X, PDB: 1PBX). Bottom right: Pol III α subunit (class C, PDB:
2HNH). The palm domain is yellow, the thumb domain is red and the fingers domain is blue.
The top two polymerases have an exonuclease domain (pale turquoise). These domains are similar in structure but have strikingly different positions in the molecules. Some of the polymerases have further domains, shown here in gray.

10. 6.1.2 DNA polymerase
Classes C and X have a different topology of the palm domain and order of the domains.
FIGURE 6.5 Top left: The palm domain in DNA polymerases has a conserved fold of the type
double split β-α-β found in many nucleic acid binding proteins. Pol I (top middle) and RB69
polymerase (top right) have a different topology from that of Pol β (bottom left) and Pol III
(bottom right). The three aspartate or glutamate residues involved in binding of the catalytic
magnesium ions are shown.

11. 6.1.2 DNA polymerase
When DNA molecules bind to polymerase, main structure conformation is changing.

12. 6.1.2 DNA polymerase
When a correct nucleotide binds, the conformational change take place generating the proper configuration in the active center.

13. Sliding clamp help the efficient processivity
6.1.2 DNA polymerase
In replication, the polymerase needs to repeat the same function thousands of times.
Sliding clamp help the efficient processivity
FIGURE 6.8 A sliding clamp: human PCNA (proliferating cell nuclear antigen), that encloses the newly replicated DNA (PDB: 1U7B).

14. 6.1.3 Telomerase
FIGURE 6.9 Replication of a piece of DNA with three origins of replication (O). In the forward direction from an origin of replication, the leading strand will be copied continuously. The lagging strand has to be synthesized as fragments. This causes a problem close to the ends, and the 5’ ends of the new DNA strands (darker color) could stay incomplete. The telomerase will assist to complete the ends of the newly synthesized strands.

15. 6.1.3 Telomerase
FIGURE The proposed secondary structure of telomerase RNA (TER) from (a) a ciliate, Tetrahymena thermophila, (b) Homo sapiens and (c) yeast Saccharomyces cerevisiae. The template is shown in cyan. (Reprinted with permission from Theimer CA, Feigon J. (2006) Structure and function of telomerase RNA. Curr Op Struct Biol 16: 307–318. Copyright (2006) Elsevier.)

16. 6.1.3.1 the telomerase holoenzyme
Overall structure is “right hand” model.
Have to N-terminal domains involved in DNA and RNA binding.
FIGURE Top: The pseudoknot from human TER. The structure contains nucleotides from stems p3 and p2b (see Fig. 6.10). The nucleotides are colored blue (guanine), green (cytosine), yellow (adenine) and red (uracil). Two nucleotides that were used in the construction of the RNA molecule are shown in gray. The central part of the molecule has a triple-helical structure (PDB: 1YMO). Bottom left: The TEN N-terminal domain of T. thermophila TERT. This domain has a unique fold and is involved in DNA binding (PDB: 2B2A). Bottom right: The RNA-binding domain of TERT from T. thermophila, which has no strong similarity to other proteins (PDB: 2R4G).

17. 6.2.1 DNA-repairing glycosylase
DNA repair system
1. Recognizing oxo-dGTP and hydrolyzing it into oxo-dGMP.
2. recognize modified base and hydrolyze the glycosidic bond.696
FIGURE Top: The deamination of cytosine (left) generates uracil (right). Bottom: The oxidation of guanine (left) leads to oxoguanine (right).

18. 6.2.1 DNA-repairing glycosylase
FIGURE The structure of the DNA repair enzyme oxoG-DNA glycosylase bound to DNA. The oxoG nucleotide has moved out of the double helix and up into the active site of the enzyme. It and its base pair partner, a cytosine, are shown in color and in thicker lines (PDB: 1EBM).

19. 6.2.2 Recombination
DNA pairing and strand-exchange are the central activities during processes involving homologous recombination.
In bacteria, RecA is a central enzyme. In eukaryote, Rad51 performs the same function.

20. 6.2.2 Recombination
FIGURE Top left: The structure of RecA from E. coli. The core domain is shown in green and the terminal smaller domains are shown in blue (N-terminal domain) and orange (C-terminal domain). The core domain is the prototype of the RecA fold found in many proteins. A bound ADP is also shown (PDB: 1REA). Top right: The human Rad51 protein has the same topology as the central sheet but the N-terminal domain is different (PDB: 1SZP). Below: A filament of Rad51, based on the crystal structure. It has six subunits per turn. The protein-protein interactions in this model fit reasonably well with functional studies. The DNA binding is not yet
structurally examined, but must be within the grooves of the filament.