DNA Repair 5: The final chapter

Another method of DSB repair is via non-homologous end joining (NHEJ), which seems significantly riskier for the cell because it involves the ligation of two ends of DNA.  Nonetheless, it appears that this is the preferred mode of double-stranded breaks and is not random.  Hypotheses have been considered that the proteins involved in NHEJ are very abundant in the cell, and when DSB occur, they are immediately available to attach to the DNA and ligate it back together.  This process is performed by Ku heterodimers, which are proteins that bind directly to the ends at the DSBs.  Ku proteins then recruit DNA-PKcs (DNA protein kinases), which, in turn, recruit Artemis endonuclease to open the ends and remove any hairpin structures that may have developed.  The ends of the DNA are then slightly chewed away, polymerized, phosphorylated, and ligated together.  The action of XRCC4, another protein that complexes during NHEJ, and Ku is used to recruit DNA ligase IV.  The final product of this process is a DNA strand that is very similar to the original DNA, but it is not exact:  NHEJ does result in mutated DNA sequences.

An excellent review on DSBs and their repair can be found here.

Double-stranded breaks sound like a terrible thing to have happen to a cell.  After all, when DNA breaks and is repaired, there is a high chance that the mutation that arises will be harmful to the cell.  Nonetheless, double-stranded breaks aren’t detrimental to the cell, and they are even required for certain normal cellular processes.  For example, in the development of immunoglobulins, lymphocytes’ genomes are rearranged and parts are removed in a process termed VDJ recombination.  This allows for the lymphocytes to increase diversity in the coding regions of the immunoglobulin genes and helps to protect us from disease.  To begin VDJ recombination, site-specific endonucleases RAG-1 and RAG-2 induce DSBs and repair these breaks via NHEJ.

As mentioned above, Ku proteins bind directly to DSBs when they occur, but how do they know when a DSB occurs?  The detection of these breaks is performed by the MRN complex composed of Mre1, Nbs1, and Rad50.  Mre11 acts as an endonuclease, Rad50 as an ATPase, and Nbs1 as a scaffold.  These proteins, once in complex, hold the DNA together and recruit other proteins, resulting in several signaling cascades.  These signaling cascades have been studied in detail using ionizing radiation induced foci (IRIF) in vitro.  The data that has been gathered from these studies shows that the MRN complex recruits ATM, a PI3K-family kinase, that becomes active during DSB formation.  ATM then phosphorylates H2AX, an H2A variant, to form γH2AX.  γH2AX acts as a scaffold to recruit more ATM and many other proteins, such as chromatin remodeling complexes, to the site of the lesion.  The cell cycle is arrested via phosphorylation of Chk2 by ATM (or phosphorylation of Chk1 by ATR – ATM-related kinase).  Chk1 and Chk2 phosphorylate CDC25 phosphatases and inactivate them, which prevents the activation of cyclin-dependent kinases.  Additionally, ATM can induce phosphorylation and activation of p53, leading to G1/S arrest.  Tip60 is another protein involved in DNA repair and is involved in acetylating ATM to activate it.  ATM and Tip60 exist in a constitutive complex that is only activated on DNA damage signaling.  MCD1 acts as a scaffold that binds phosphorylated H2AX and docks ATM/Tip60.

DSB detection simplified:

1. The MRN complex recognizes and binds the free ends of the DNA
2. ATM is recruited to the foci
3. ATM phosphorylates H2AX, Chk2 and p53
4. Chk2 phosphorylates CDC25
5. Cell cycle is arrested and chromatin opens for DNA repair

Several diseases are associated with defects in the DSB response pathway.  Among them are ataxia-telangiectasia, resulting from mutations in ATM.  Several mutations in ATM will result in the A-T phenotype, and approximately 1% of the population carries one mutated allele.  Those that suffer from this syndrome show immunodeficiency and increased cancer rates, and they have a life expectancy of 17-23 years.  In addition to A-T, Nijimegen breakage syndrome results from mutations in Nbs1, and A-T like disorder (ATLD) results from Mre11 mutations.  Breast cancer may also have connections to DSB repair, as BRCA1 is associated with the BASC complex, which is involved in this and many other pathways for DNA repair.  BRCA1 acts as an ATM substrate and localizes to IRIFs, possibly acting as a scaffold.

More information about ataxia-telangiectasia can be found here.

On NBS can be found here.

A paper about ATLD: here.

Identification of genes involved in DNA repair

A number fo methods have been developed that specifically scan organisms in order to detect defects in DNA repair.  Isolation of the affected gene and protein then allows for recognition of specific DNA repair proteins.  Specifically, one can look at the frequency of mutations (see Ames test, for example) as well as elevated sensitivity to ultra-violet or ionizing radiation.  Cell cycle arrest (cell division defects) and apoptosis are common phenotypes associated with defective enzymes as well.

DNA Repair 4

A further method of repairing small lesions in the DNA is via mismatch repair (MMR), which fixes errors in replication and heteroduplexes from recombination intermediates.  The process involves the Mut proteins in prokaryotes.  MutS bound to ATP recognizes and binds the unpaired base to activate the mismatch repair enzymes.  MutH is involved in determining which DNA strand is methylated, indicating the parent strand (the correct copy of the DNA that should be used to repolymerize the incorrect copy).  MutL facilitates in assembling the MMR complex, which consists of helicases and exonucleases.  Repair is then done by DNA polymerase and ligase, and methylation of the newly copied (and unmutated) strand is then performed.  MutL and MutS homologues are found in both yeast and humans, with varying roles and functioning as heterodimers.  Additionally, in eukaryotic MMR, the ability of the enzymes to distinguish between parent and daughter strands is not as clear.  Hypotheses have emerged that consider interaction with PCNA and identification of nicked strands (on the nascent DNA) as mechanisms that may assist in determining which strand needs to be repaired.

 

One consequence of an impaired mismatch repair system is microsatellite instability, a variation in tandem repeat sequences.  In cells both with and without a functioning MMR system, changes in the number of repeats is commonly observed as the DNA is being replicated.  This variation is due to slippage of DNA polymerase as it runs through repeated sequences, as if it has gotten lost along the way.  Usually, the microsatellite instability is held in check by MMR enzymes that are able to recognize the bubble that forms in the DNA.  However, in those individuals with mutations, there can be a vast array of repeat numbers in these microsatellites, up to 3.3x10^-3 mutations per cell per generation.   

The most catastrophic event that can occur in a cell is double-stranded breaks (DSBs) in the DNA, which can be caused by radiation and mutagens, but it can also result from DNA replication and physiological processes (harmful metabolites that are not controlled by the cell properly).  Two methods have evolved to prevent these DSBs from having a negative impact on the cell, which we will consider now.

The first method of DSB repair is by homologous recombination.  (Details about this pathway can be found in additional posts about recombination.)  There are two methods for recombination: synthesis-dependent strand annealing and single-strand annealing.  In synthesis-dependent strand annealing homologous recombination, strand invasion of the undamaged DNA (sister chromosome) by the DNA that contains the double-stranded break.  New DNA is synthesized from the correct copy and ligated to remove the DSB.  In contrast to synthesis-dependent annealing, single-strand annealing relies on an uneven breakage in the DNA.  Several bases on each side of the DNA strand are removed, and the staggered ends are annealed to create fixed DNA.  This method of DSB repair is especially common in stretches of repeated-sequence DNA.

DNA Repair 2

Avoiding Permanent DNA Damage

When a cell encounters DNA damage, it has a number of responses that help to correct any errors and propagating these errors to progeny cells.  Thus, the cell devotes a number of enzymes to detecting and repairing any lesions in the DNA.  Correcting DNA lesions is more important than fixing errors in transcription or translation because of the relative lifespans of the DNA versus mRNA and protein:  mRNA and protein are both expressed and, while they can be longlived, they are not passed on to progeny.  In contrast, DNA of daughter cells is an exact copy of the mother cell, and these daughter cells will carry any lesions that the mother cell carried as well.  Therefore, to prevent long-lasting mistakes in the DNA, the cell fixes any errors that it may encounter.

One of the first hurdles that the cell encounters when it must maintain its genome is that it must accurately copy it, but it must also perform polymerization of DNA quickly enough to facilitate survival.  Once in a while, DNA polymerase will make a mistake, roughly one in a billion base pairs is misincorporated, which is an astoundingly low rate.  Such a low mutation rate is thanks, in part, to the 3’-to-5’ exonuclease activity of DNA polymerase.  When the enzyme is polymerizing DNA and it accidentally adds the wrong nucleotide, this mistake is detected because DNA polymerase is not able to polymerize efficiently from a misincorporated nucleotide.  When the mistake is detected, the exonucleolytic activity of DNA polymerase kicks in and removes several nucleotides behind the incorrect base.  Then, it reverts to its normal function and continues to replicate the DNA. 

When DNA polymerase δ is replicating the DNA and there is an identified lesion ahead of the replication fork, the cell can choose to bypass the lesion and repair it later.  Such a scenario is called translesion synthesis, which uses an alternate polymerase and is not high fidelity, as DNA polymerase δ.  The process involves the ubiquitination of PCNA (proliferating cell nuclear antigen) and the involvement of a number of factors normally involved in DNA repair, including RAD6 proteins and DNA polymerase η.  More information about this alternative mechanism can be found here. 

DNA Repair 1

Causes of DNA Damage

Constantly, day-in and day-out, the genome is assaulted by a number of forces that lead to damage and mistakes that can lead to severe problems for the organism under the onslaught.  Included in the forces that cause the DNA damage are chemicals, radiation, and especially reactive metabolites (reactive oxygen species, for example).  Such mutagenic agents can lead to damage in the form of thymidine dimers, single-strand breaks, creation of abasic sites, or the formation of double-stranded breaks.  Specifically, UV radiation induces pyrimidine dimers, while ionizing radiation can cause double-stranded breaks.  Toxic chemicals can be mutagenic in themselves, or they can be activated by cellular metabolism into mutagenic chemicals.  In addition, DNA replication errors and the inherent mobile genetic elements can lead to genetic mutation.

Some of the most common mutations in DNA occur via hydrolysis a nucleotide base.  Due to the high concentration of water within the cell, the DNA is constantly exposed to the harmful effects of water.  Depurination results in a nucleotide without its guanine or adenine base.  Deamination is a base conversion event in which a methylated cytosine residue to converted to thymine.  Such an event can be especially dangerous to the cell because it can induce base changes if the mutation is not fixed immediately. 

Chemicals that induce mutations come in several forms: base analogs, intercalating agents, and base modifiers.  In the case of base analogs, the chemicals resemble that of the natural bases found in the genome but have altered binding potential, leading to the misincorporation of bases that fit improperly.  Examples of base analogs include 5-bromouracil (5-BU) and 2-aminopurine (2-AP).  Intercalating agents do not pair with the DNA bases, but they affect the structure of the DNA helix during DNA synthesis, leading to improper pairing and the induction of mutations.  Examples of intercalating agents include proflavin and acridine orange.  When a base is modified, it can also affect the DNA structure, leading to altered pairing and the induction of mutations.  Chemicals that modify bases as alkylating agents include ethyl methanesulfonate (EMS) and nitrosoguanidine (NG). 

Testing for mutagenic activity

In order to examine whether a chemical is mutagenic in a living system, using the model organism Salmonella, the Ames test is employed.  First, mice are exposed to the mutagen and injected with arochlor to induce hepatic function, where most chemical mutagens are activated.  Then, liver extract is homogenized and added to Salmonella.  The Salmonella strain used contains a His- marker, making it unable to grow in the absence of histidine.  The ability for the strain to grow on media lacking histidine works as a readout for the mutagenic capability of the chemical:  the most colonies that grow on the selective media, the more mutants were able to convert to His⁺ and the higher the mutagenic capacity of the tested chemical.  Extensions of the Ames test have been developed to specifically look at base substitutions and insertions or deletions.

The Ames Test Simplified:

1. Expose mouse to potential mutagen
2. Inject mouse with arochlor to induce hepatic function
3. Isolate and homogenize liver
4. Combine homogenized liver with Salmonella typhimurium (His^-)
5. Plate Salmonella on agar lacking histidine
6. Count number of colonies that have grown on the plate as a measure of mutagenicity.

More information about the Ames test can be found here and here.

Note: Links provided in this post are only a sampling of the vast amount of information available about these chemicals and processes.