Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

DNA Replication and Repair
Replication fork

For a DNA duplex to function as a template during Replication, its two strands must be unwound (also referred to as melted) to provide access to the nucleotide bases of the dNTPs from which the DNA polymerase builds the daughter strand.

This unwinding is driven by helicase Enzymes utilizing the energy of ATP Hydrolysis.

Unwinding begins at specific DNA sequences known as replication origins, or simply origins. Nucleotide sequences at origins vary widely across different organisms, although they share a common feature: an elevated concentration of A-T Base Pairs in these regions. Such areas contain fewer Hydrogen Bonds between the DNA strands, making DNA melting (unwinding) easier.

Once the helicase unwinds the duplex, a specialized enzyme called primase (a specialized RNA polymerase) synthesizes a short RNA primer complementary to the unwound region of the template strand. This DNA-annealed primer is then extended by DNA polymerase to form the daughter strand.

The region of DNA where all these enzymes assemble to synthesize the daughter strands is called the replication fork, or growth fork.

During replication, the replication fork and its associated Proteins move away from the origin, which leads to a buildup of torsional stress in the DNA. This stress is relieved by the enzyme topoisomerase I. For the replication process to proceed continuously and allow DNA polymerase to travel along and copy the DNA duplex, the helicase must continually unwind the duplex while the topoisomerase removes the resulting supercoils.

The main challenge in "handling" DNA at the replication fork stems from two factors:

1) the two parental DNA strands are antiparallel,

2) DNA polymerase (much like RNA polymerase) catalyzes the growth of the daughter chain exclusively in the 5'→3' direction.

Synthesis of the daughter strand, known as the leading strand, can proceed continuously, starting from a single primer in the 5'→3' direction—that is, in the same direction in which the replication fork moves (Figure 53).

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Figure 53 - Schematic of leading and lagging strand formation

Complications arise during the synthesis of the other daughter strand, known as the lagging strand.

Since the growth of the lagging strand must occur in the 5'→3' direction, template copying must proceed in the direction opposite to the movement of the replication fork.

The Cell solves this problem by synthesizing new primers every few hundred bases on the template strand as the duplex unwinds and progressively more bases are exposed.

Each of these primers, annealed to the template strand, is extended in the 5'→3' direction to form continuous segments known as Okazaki fragments (named after Reiji Okazaki, who discovered them).

The RNA primer of each Okazaki fragment is removed and replaced with DNA by the adjacent growing Okazaki fragment. The enzyme DNA ligase then seals the neighboring fragments together.

Detailed insights into how proteins participate in eukaryotic METABOLISM/36.html">DNA replication were gained by studying the replication of small viral DNAs, particularly the circular DNA of the simian virus SV40 (Figure 54).

Figure 54 - Schematic of the SV40 DNA replication fork and associated proteins: a - replication fork; b - schematic of PCNA encircling the DNA duplex; c - schematic of RPA positioning relative to the DNA template strand

Figure 54 illustrates the proteins that coordinate SV40 DNA copying at the replication fork. This multi-component complex enables the cell to execute an orderly sequence of events that carry out essential cellular Functions.

Within the molecular "machine" that replicates SV40 DNA, a hexamer of the viral protein known as the large T-antigen acts as a helicase, unwinding the parental helix at the replication fork.

All other proteins involved in replication are native Proteins of the infected cell.

Primers for both the leading and lagging DNA daughter strands are synthesized by a complex (primase-Pol α). In this complex, primase, which synthesizes the short RNA primer, works in tandem with DNA polymerase α (Pol α), which extends the RNA primer with deoxynucleotides to form a hybrid RNA-DNA primer.

Subsequently, this primer is extended into a daughter DNA strand by the enzyme DNA polymerase δ (Pol δ), which copies the template strand with higher fidelity and fewer errors than Pol α. In literature, DNA polymerase δ is also referred to as DNA polymerase III.

Pol δ forms a complex (PCNA-Rfc-Pol δ) with replication factor C (Rfc) and proliferating cell nuclear antigen (PCNA), which replaces the (primase-Pol α) complex following primase action.

The homotrimeric PCNA protein features a central channel through which the daughter DNA duplex passes (Figure 54(6)). In doing so, PCNA prevents the dissociation of the (PCNA-Rfc-Pol δ) complex from the template strand.

Once the parental DNA at the replication fork has unwound into two template strands, heterotrimeric RPA (replication protein A) proteins bind to these strands (Figure 54(b)). RPA proteins belong to the class of single-strand binding proteins, also known as helix-destabilizing proteins.

Binding of RPA maintains the optimal conformation of the template strand for DNA polymerase-mediated replication. These RPA proteins are displaced from the template strand by Pol α and Pol δ as they synthesize complementary strands paired with the parental strands.

Some eukaryotic proteins involved in DNA replication are not shown in Figure 54.

The enzyme topoisomerase I binds to the parental DNA ahead of the helicase to relieve the torsional stress generated during unwinding of the helix.

Specialized enzymes, Ribonuclease H and FEN I protein, remove the ribonucleotides at the 5'-end of Okazaki fragments. These ribonucleotides are replaced with deoxyribonucleotides by DNA polymerase Pol δ upon reaching the adjacent Okazaki fragment.

The completed Okazaki fragments are joined together by DNA ligase, an enzyme that catalyzes The formation of the standard 5'–3' phosphodiester bond.

During DNA Synthesis, DNA polymerase makes an error approximately once every 10,000 steps, incorporating an incorrect base into the growing DNA strand. The Human Genome contains 3x10⁹ base pairs, and without proofreading and error correction mechanisms, the mutation rate would be catastrophic. Fortunately, DNA polymerase features a built-in proofreading mechanism to detect and correct errors.

DNA polymerase contains two active sites.

The first Active Site monitors the spatial arrangement and template complementarity of each incoming nucleotide, catalyzing the Formation of the sugar-phosphate bond between the new nucleotide and the newly synthesized DNA strand.

Following the action of the first active site, the second active site pulls on this terminal nucleic acid base, attempting to dissociate it from the newly synthesized double-stranded DNA. If the bond of the new base is insufficiently strong, the second active site excises and removes it. This additional checkpoint prevents mismatched base pairing and increases The fidelity of template-directed synthesis by orders of magnitude.