How Do DNA Repair Enzymes Work?
It’s an absolute truth that human beings have the ability to make mistakes in their DNA synthesis. DNA repair enzymes are cells’ corrective mechanisms. James Stivers, a bioengineer and expert in quality control of billions of widgets every day, has dissected the movement of one of these enzymes. In this fascinating and thought-provoking video, he explains how the enzyme works.
Base excision repair
The base excision repair (BER) pathway is a DNA damage-repair pathway that maintains genomic integrity by removing abnormal or damaged bases. BER is made up of four or five steps, including DNA glycosylase, AP-endonuclease, DNA polymerase, and ligase. Single-strand breaks in DNA created by free radical reactions always have block termini. Although BER is one of the simplest repair pathways, its functional significance is still poorly understood.
BER proteins work together in concert to recognize damaged bases. DNA excision repair enzymes are grouped in temporally controlled complexes called BER proteins. BER proteins cross-talk with DNA to ensure that damaged base recognition is accomplished with maximum efficiency. These enzymes are involved in a complex process called DNA repair. Ames BN, Beckman KB, and DeMott MS studied BER proteins in yeast.
APE is a protein responsible for base excision repair in bacterial cells. It is a member of the SF2 ATPase family. It is active in mitochondrial DNA and bacterial cells. It recruits the Uvr (A)BC nucleotide excision repair machinery by interacting with the abasic-site-containing DNA strand. The activity of aPE is dependent on the presence of a base-specific protein called aprataxin.
Glycosylases are another type of DNA repair enzyme. They recognize damaged bases and flip them out of the major groove. They are damage-specific and exploit the deformability of DNA at the base pair. BER involves two types of end-processing enzymes. One, Apn1p, and Rad2p, are bifunctional. Each of them excises a damaged base and cleans up the DNA strand.
The genes coding for NER is found in many organisms, including humans. Human eukaryotes have more complex NERs than prokaryotes. Mammalian cells contain nine different NER proteins. The protein names are related to the disease. For example, the XPA protein is responsible for xeroderma pigmentosum disease, while XPC and CSA are associated with the Cockayne syndrome.
Mismatch repair is an important biological process. In bacteria, DNA molecules that contain mismatches induce a repair reaction. MutS, MutL, and MutH are implicated in the process. Inactivation of any of these genes increases the mutation rate fifty to one hundredfold. Other bacteria such as Saccharomyces cerevisiae have demonstrated the existence of MMR enzymes and elementary mechanisms.
Eukaryotic mutL homologs include MutSa and MutSb and PMS1 and PMS2. Humans have a homolog of MutLa called MLH1, which recruits other molecules to the complex. MutLa initiates the endonuclease activity of PMS2, which then opens the entry site for exonuclease 1. Both of these enzymes are essential for mismatch repair.
The Msh1 protein, a precursor to PCNA, is required for DNA mismatch repair in humans. These proteins have diverse roles in DNA replication. These enzymes are associated with various other genes in the cell, including PCNA. Their roles in DNA replication are discussed below. DNA mismatch repair is an important biological process essential for cell survival. A variety of genes in our bodies are implicated in mismatch repair.
In addition to MMR, other important pathways in our body maintain genomic stability. Deficiencies in mismatch repair cause cancers and microsatellite instability—a cell’s mutation rate increases when it fails to repair errors in DNA replication. Microsatellite instability, which occurs when microsatellites vary in length, is a hallmark of tumor cells. If a cancer cell fails to repair the mismatches correctly, it will undergo a high mutation rate and can lead to tumors.
The mechanism of protein-DNA interactions is remarkably dynamic. The dynamic exchange model facilitated dissociation and explains the increased kcat values for many BER enzymes in their reactions with their downstream enzyme. In addition, the steady-state turnover number (kcat) of DNA repair enzymes is thought to be a readout for coordinated activity. This processive searching mechanism may help explain the high variability observed in the kcat values of DNA repair enzymes.
One of the key properties of AlkD DNA repair enzymes is their ability to dissociate into a bulk solution without the help of an enzyme downstream. If an enzyme is incapable of interacting with DNA adjacent to its product, it will most likely dissociate into the bulk solution by nonspecific binding or direct dissociation. In addition, the enzymes’ specificity towards damaged DNA sites will need to be carefully studied before they can be harnessed as therapeutic agents.
The aging process and other factors can lead to DNA damage, which eventually overtakes the repair rate. The resulting accumulated errors lead to apoptosis, senescence, and cancer. In addition, faulty DNA repair has been linked to inherited diseases, leading to premature aging and increased sensitivity to cancer. However, a lack of DNA repair enzymes does not necessarily mean a death sentence, as overexpression of DNA repair enzymes can increase lifespan and resistance to carcinogens.
AL reduces the expression of DNA repair enzymes mRNA and reduces the efficiency of nucleobase excision in animal models. The results show that the shielding effect of AL may be due to an additional DNA repair mechanism. The increase in mRNA expression of these enzymes and their activity is also associated with an increase in the excision of lesioned nucleobases. Eight-oxoG exhibited the highest repair activity in the AL-treated group, suggesting that AL may play an important role in preventing and minimizing DNA damage.
Despite the widespread use of DNA repair genes to improve genome function, the relationship between the levels of mutations and GC content in the proteome has not been fully clarified. While genomes lacking mutT+ DNA repair enzymes have smaller GC content than genomes with all four genes, they significantly correlate positively with proteome size. However, the lack of DNA repair genes in the genomes of pathogenic bacteria cannot be attributed solely to the absence of mutL or mutS.
The mutL gene product recruits the complex and identifies the mismatched base pair on the duplex DNA strand. The mutH gene product recognizes a mismatched base pair on a DNA strand during this process. Afterward, MutT+ DNA repair enzymes bind to the hemimethylated motif and cleave the DNA strand. A nick is left on the strand containing the unmethylated GATC.
Mismatch repair is highly conserved in evolution and explains why certain mutations are linked to cancer. In addition to bacterial species, various mammals and other species have homologs of E. coli genes. Analysis of hereditary nonpolyposis colon cancer (HNPCC) mutations revealed that these proteins encode identical amino acid sequences. Furthermore, these proteins are present in human DNA and are termed human mutL and mutS homologs.
MUTYH, a well-known oxidative DNA repair enzyme, initiates base excision repair by recognizing A:8-oxoG mismatches. MutY homologs accumulate 8-oxoG in DNA and are associated with an increased risk of carcinogenesis. HK-2 cells express high levels of MUTYH and TGF-b1, which triggers EMT. In contrast, H2O2 did not affect MUTYH expression. These inconsistencies pointed to mechanisms other than oxidative DNA damage.
UvrA2 recognizes the damaged region and forms a dynamic complex with another protein called UvrB. These two proteins then make two endonucleolytic cuts on the damaged strand. UvrB then forms a complex with UvrC, which is a nuclease. It incites both sides of the damaged DNA segment and directs synthesis. After the repair process is complete, the damaged segment is no longer damaged.