Some Eukaryotic DNA Repair Systems

It is in the best interests of a cell (or of an entire multicelled organism) to prevent cells with DNA damage from undergoing DNA replication (S phase). Arrest in G1 phase would give more time for repair of the damage, as well as prevent the possible mutations that might arise from attempting to replicate injured DNA, and might even prevent cell death from the attempt to replicate improperly prepared DNA. Cell cycle arrest may be triggered by many insults to the DNA structure, but recent studies with Ionizing radiation (IR) make it reasonable to look at this aspect of DNA damage.
Three of the major actors in cell cycle arrest are: the p53 gene (a cell cycle "checkpoint") and its product, which is a major player in the cell cycle response to cellular insult, and is involved in spindle formation, cell growth and apoptosis; IRF-1 (interferon regulatory factor 1), which is a factor which is normaly known to act on interferon to suppress cell growth and tumor activity; and c-Abl, an enzyme that phosphorylates other proteins. These factors seem to act independently of one another, but may use some of the same factors and pathways in their actions. Some of these will be looked at in greater detail below.

IR induces IRF-1 (but without transcription - activation from a bound or inactive form?). The IRF-1 binds to the promoter region of the p21 gene (a known inhibitor or the cell cycle) and up regulates its transcription, so that the p21 gene product is formed. p21 then inhibits CDKs, especially CDK 2 and CDK 4 (major actors in the G1 to S phase transition), and interacts with (and inhibits) PCNA (needed for DNA replication - somewhat analogous to the bacterial beta factor), thus blocking the phase transition and DNA replication.
IR induces p53, whose gene product binds to the promoter (but in a different site than IRF-1) region of the p21 gene, and up regulates it. Cycle arrest proceeds as above.
IR (or other damaging agents) induces the c-abl gene, which produces c-Abl kinase. The kinase complexes with p53 and aids in inactivating CDK 2, by some unknown mechanism (not excluding activating transcription of some gene, but not involving p21, since the activation of p21 - which c-Abl does aid - does not lower CDK 2 activity - in contrast to the above).
In addition to these actions, cAbl binds Rb protein and this complex is involved in G1 to S arrest at a later point in the cell cycle. If it is already past S phase, the cell must still be blocked form dividing before repairs are made.

Another mechanism utalizes an SOS - like response, where a set of genes, inactivated bt the Crt protein (which is phosphorylated - and inactivated - in response to ssDNA) lead to repair of DNA and stoping the cell cycle in G2.

Yet another mechanism is controlled by CHK kinase (Xchk1 in Xenopus). ATR (also known as Me and Rad3 in other systems) uses claspin (a DNA associated Protein which binds CHK1) to activate CHK1 by phosphorylation at multiple sites.
Claspin, itself, binds to chromosomal DNA by interacting with the prereplication complex (pre RC) Cdc45 and Cdk2, so it might be bound near replication forks.
Activated CHK1 blocks activation of the Cdc2-cyclin B complex (MPF) by inhibiting cdc 25 and stimulating Wee 1.
After repair, the Chk1 is phosphorylated by another regulatory protein (Crb2 in yeast), by cdc2.
In some cases, when the damage can't be repaired, the cell is allowed to go through division anyway, in the hope that repairs might be made in subsequent cell cycles. This process is known as Adaptation, and is known in singel cell and at least one multicell (Xenopus) creature. In this circumstance, claspin is inactivated by phosphorylation by a "polo-like-kinase" (Plk; plx in Xenopus). Other examples of adaptation seem to involve the inactivation of the checkpoint kinase Rad53 by a Plk.

DNA Repair Systems

The repair mechanisms use different mechanisms of the expanding DNA polymerase family, some of which have prokaryotic homologues, as do many other factors in these systems, such as repair of DNA methylation by ABH2 and ABH3, eukaryotic single and double strand alkylation repair homologues of bacterial AlkB.

One question is whether new histones are added after a repair, or if old ones are recycled. Recently, it has been found that histone H3.1 (at least) is added as a new molecule, not recycled. This process uses the chaperone chromatin assembly factor (CAF-1) to load the histone on.

Postreplication Repair

This type of activity repairs damage due to errors or mutational events, and is most effective on small gaps and mispairings. The major actors are hHR6A and hHR6B, which form an active complex. Mutations in these genes lead to less efficient repair. HHR6B, especially, is involved in the adding of Ubiquitin (UBC) to histones H2A and H2B, a process implicated in chromosome condensation, and so is found in conjunction with chromatin under most conditions. Loss of function of the equivalent of hHR6B leads to male infertility in mice (lack of proper spermatogenesis). Some of these systems use human pol ß 2 (pol lamda)

Mismatch Repair

There are many similarities between prokaryotic and eukaryotic mismatch repair systems. The MutSH2 /MutSH6 (MutSα) or Mut SH3 and Mut SH3 (MutSβ) heterodimer binds to the mismatch, recruits the MLH1 and PMS2 (which contains endonuclease activity) heterodimer (MutLα) and use ATP to move along (?) the DNA until a nick is located (between Okazaki fragments) or use MutLα to create a nick, (if necessary, above thmismatch), on which EXO 1 can act in a 5' to 3' direction, followed by RPA, Polδ with PCNA/RFC and Ligase 1. This can happen even if the mismatch is hundreds of bases away from the nick. MutLH1 also forms heterodimers with other factors, one of which, the MUtLH1 - MutLH3 - also with endonucleic activity - (MutLγ) is involved in recombination, which maight play some role in longer repairs. Mishmatch repair seems to be coordinated with DNA replication. Exactly how the system decides which is the old and which is the new strand is unclear, unless one assumes that there are single strand nicks in the new strand.

Nucleotide Excision Repair

Activity: uv (or other mutagen) induces damage. XPC binds hHR23b (not needed for activity) and TFIIH, to begin assembly of the repair complex. XPA complexes with XPF and ERCC1, which binds at the damaged area. XPG binds TFIIH (and also PCNA), and the factor is complete. XPF and XPG probably provide the nicks at either end of the damage site, and XPB and XPD probably act as repair helicases, cooperating in opposite directions to open up the DNA for repair. Repair is probably analogous to the prokaryotic system. TFIIE, another transcription complex subunit, inhibits the helicase activity of XPB, by binding to TFIIH. TFIIH is not usually found associated with the transcription complex during elongation, so its effects in repair (and transcription) probably occur initially at the promoter region of the gene (and initiation of transcription). A related system, transcription coupled repair (TCR), uses many of the same factors, which may be recruited by CSA and CSB. The repair is then completed when the endonucleases XPF and XPG nick on either side of the damage (24 - 32 bp insize) and PCNA, pol delta or epsilon and ligase repair the lesion.

Other error - prone repairs can be completed by specific systems, such as those using Polymerase κ (kappa) which can bypass an abasic site by skipping the missing base and creating a frameshift mutation (like prokaryotic pol IV), and polymerase ι (iota), which often puts a G (instead of an A) opposite a T. The usual effect in abasic substitution is to insert an A opposite a missing base, but others are also possible. The factors usually involved are the REV 1, 3 and 7 proteins involved in pol ξ (xi).

UV Damage Repair

Rev1 inserts a C opposite an abasic site, but needs polymerase ξ (xi) to extend the repair farther.

DNA polymerase η (eta) is involved in reading past T-T dimers. Damage in systems relating to this polymerase result in the disease Xeroderma pigmentosum. In these cases, possibly the Rev 1 & 7 dimer (yeast polymerase ξ) takes over, with extermely error - prone repairs.

Single Strand Break Repair (SSBR)

SSBRs can come from direct chemical action (free radicals, etc.) or as the result of enzymatic actions (Base excision repair, etc.). The same general four step pathway is used to fix both types of damage. The pathways may be coordinated through a central scaffolding protein, XRCC1, which is known to interact with many of the enzymes and complexes in the pathways (APE,PARP,PNK, DNA ligase, DNA polymerase β).

1.    Bind to the damaged DNA region.
                     a.  Many DNA repair systems use AP endonuclease (APE) to excise the damaged region and can "pass off" the repair to the next repair subunit and so can be protected from further problems (recombination) or damage.
                     b.  Lesions due to chemical action need to be detected. Poly(ADP-ribose) polymerases PARP-1 and PARP-2 can do this. They can recruit other components, make ATP for enzymatic repairs, such as DNA ligase, and inhibit recombination.

2.    End processing - most damaged or partially repaired DNAs have modified 3' or 5' ends (indeed, APE itself adds an extra 5' deoxyribose phosphate (dRP) to DNA that it cuts). There are different ways of handling these problems. DNA polymerase β can remove 5' dRP after it fills in the ss gap. APE 1 can remove 3' - P or 3' - sugar groups (even though it adds 5' dRPs) when helped by XRCC1 (a central DNA repair complex). XRCC 1 can also recruit DNA polynucleotide kinase (PNK), which can fix both 3' and 5' problems. XRCC1 is inactive until it is phosphorylated by CK2, which has to act during or just after the damage is done in order to get proper repairs. CK2 has about 300 substrates, of which XRCC1 is one. CK2 has two catalytic subunits (α and/or α'), and two controlling subunits (β₂). CDK2 is deregulated in many tumors. CDK2 phosphorylation of XRCC1 seems to have its largest influence on the XRCC1 - PNK interaction, but it also influences the other interactions.

3.    Gap filling - Usually use DNA pol β to add one base, but it can add up to about 15 (short pacth repair). FEN-1 can be used to get rid of the polynucleotide "flap". In long patch repair, pol δ and ε probably are used.

4.    Ligation - DNA ligase IIIα (lig 3α) is probably used for short patch repair, and DNA ligase I (lig 1) is probably used for long patch repair.

Related systems repair damage due to topoisomerases.

Double Stranded Break Repair

Homologous Recombination Repair (HR)

Acts at meiotic prophase and/or at G1 and S phases. Uses a set of genes - the Rad 51 epistasis group - which is highly conserved amongst eukaryotes, and which includes the eukaryotic RecA homologue. It may act very much like prokaryotic recombination repair. It probably interacts with the BRCA 1 and 2 gene products. There are two pathways, Synthesis - Dependent Strand Anealing and Single Strand Anealing.

Non - Homologous End Joining (NHEJ)

Acts when there is no sister chromatid available, but is also known to take place in G1. Joins may occur at short regions of homology at dsDNA ends.

Some γ radiation repair systems in mice (Mdm - 2 and Gad 45α) as well as various cyclins D1 and A) are regulated bt the mPer2 gene product, which is also involved in control of circadian rythms.

There is some indication that HR and NHEJ may share a common set of controls. Experiments on the formation of antibody genes (which routinely splice DNAs together to form new antibodies) indicate that a set of proteins (RAG 1 and RAG 2) control both types of recombination. The RAG proteins initiate recombination by nicking one DNA strand at a specific RSS site. These sites are usually paired, and after synapsis, or strand invasion, they nick again at the RSS site on the other strand, causing a DSB, which is repaired by NHEJ. Thus, it seems that RAG proteins can initiate HR by nicking and releising the DNA strand, and cause NHEJ by nicking again and conveying the DSB to the NHEJ system. RAG mutants deficient in various aspects of their nicking ability paired with DNA mutations in the RSS regions have lead to these conclusions.

Repair at Stalled Replication Forks

These are a set of 'last chance' set of methods that are used after all else fails (see above).
Some tables and figures after: http://mcbio.med.buffalo.edu/RPN530/ ; others after Nature Reviews 1:101 (2000)

If all else fails, the cell should self - distruct (apoptosis). The protein Bcl - X_L is involved in some of these dicisions

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