Question

Outline the processes involved in the following five major classes of DNA repair systems: 1. Direct Repair 2. Base Excision repair 3. Nucleotide Excision Repair 4. Mismatch Repair 5. Bypass repair systems

Answer 1

Certain chemical changes may occur in DNA spontaneously or due to exposure to chemicals or radiations. Such changes may damage the DNA and block processes like replication or transcription and can cause mutations.

Most of the damages are repaired by Direct Repair, which is the removal of faulty bases by re-synthesis of the excised region. The damage is reversed by an enzyme DNA photolyase by photo-reactivation. This enzyme uses the absorbed light to cleave carbon-carbon bond (C-C) of the pyrimidine ring of thymine dimers.

Base excision repair system involves an enzyme called N-glycosylase which recognizes the abnormal base and hydrolyzes glycosidic bond between the base and its sugar. Another enzyme, an endonuclease cleaves the DNA backbone on the 5′-side of the abnormal base. Then the DNA polymerase by its exonuclease activity removes the abnormal base. The DNA polymerase then replaces it with normal base and DNA ligase seals the region.

Nucleotide excision repair system includes three steps, incision, excision, and synthesis. The incision is done by the endonuclease enzyme precisely on either side of the damaged patch of the strand. In this way, damaged portion of the strand is cleaved. Endonuclease enzymes involved are UvrA, UvrB which recognize the damaged stretch of the strand. UvrC makes two cuts (incision) on either side. Exonuclease removes the damaged strand. The enzyme involved is UvrD. Later, DNA polymerase synthesizes the new strand by using a complementary strand as a template. DNA ligase forms phosphodiester bonds that seal the ends on newly synthesized strand.

Mismatch Base Repair: Sometimes wrong bases are incorporated during the replication process, G-T or C-A pairs are formed. The wrong base is always incorporated in the daughter strand only. Therefore in order to distinguish the two strands for the purpose of repair, the adenine bases of the template strand are labeled or tagged by methyl groups. In this way the newly replication DNA helix is hemimethylated. The excision of the wrong bases occurs in the non-methylated or daughter strand.

Bypass Repair Systems: In addition to various processes for removing lesions from the DNA, cells have developed specific mechanisms for tolerating unrepaired damage during the replication of the genome. These mechanisms are collectively called DNA damage bypass pathways. The Y family of DNA polymerases plays a key role in DNA damage bypass. Y family DNA polymerases, REV1, POLH (DNA polymerase eta), POLK (DNA polymerase kappa) and POLI (DNA polymerase iota), as well as the DNA polymerase zeta (POLZ) complex composed of REV3L and MAD2L2, are able to carry out translesion DNA synthesis (TLS) or replicative bypass of damaged bases opposite to template lesions that arrest high fidelity, highly processive replicative DNA polymerase complexes delta (POLD) and epsilon (POLE). REV1, POLH, POLK, POLI and POLZ lack 3'->5' exonuclease activity and exhibit low fidelity and weak processivity.

Example:

The Rad6 and Rad18 proteins

The Rad18 and Rad6 proteins are essential for all aspects of damage bypass. Mutations in these genes have profound effects on cellular survival after radiation damage. Sequence comparison with other proteins and biochemical studies have revealed that Rad18 is an ATPase capable of binding ss DNA. In addition, Rad18 contains a "ring finger" domain that helps it to interact with Rad6. Rad6 is a ubiquitin-conjugating enzyme—an enzyme capable of transferring ubiquitin from a ubiquitin-activating enzyme to a protein substrate. The fact that mutations in Rad6 that destroy its ubiquitylation activity also knock out the Rad6 replication bypass pathway makes it clear that the ubiquitylation function of Rad6 is essential for this pathway. One, perhaps the only essential (in this pathway) target of Rad6 is PCNA, which is mono-ubiquitinated by Rad6 on its lysine 164.