Structural Biochemistry/DNA Repair/Mismatch Repair

Introduction

Mismatch Repair in mammals is an important mechanism in the overall processes of DNA repair. Mismatch Repair (MMR) works by removing incorrect base pair match-ups in double-stranded DNA and replacing it with the correct base pair. However, MMR has other known functions, including mutagenesis in different in vivo conditions.

Canonical MMR Mechanism

Errors in DNA replication pose many problems to both the integrity of the DNA and to the individual. MMR is one way that these errors are fixed, as it is known that deficiency in MMR causes cancerous tumors in animal models.

The basic MMR system relies on the proteins, MutSα, MutLα, EXO1, RFC, PCNA, RPA, polymerase-δ, and DNA ligase I. There are three basic steps of MMR known as licensing, degradation, and resynthesis.

Licensing

In licensing, MutSα binds to the mismatch error in the DNA strand, which causes a change in the conformation of MutSα into a sliding clamp. This change is dependent upon an exchange of ADP for ATP. MutLα is recruited to forma ternary complex with MutSα, which then diffuses along the DNA strand until it reaches PCNA.

PCNA, or proliferating cell nuclear antigen, is a protein that can undergo a conformational change to become a ring around DNA. To attach to the DNA, it relies on the function of RFC, or replication factor C. This protein uses ATP hydrolysis to attach the PCNA to the DNA. This attachment is only efficient when there is a "nick" in the DNA, or an apyrimidinic (AP) site. The PCNA can then attach to the 3' end of the nick. While RFC can add PCNA without a nick in the DNA, this is down with extremely low efficiency.

Once it reaches PCNA, the cryptic endonuclease of MutLα is activated and causes additional nicks that are on both sides of the mismatch error on the same strand. This is only necessary for PCNA binding on the 3' side of the mismatch. Nicks are only made on the same strand as the mismatch because PCNA is not symmetric and has distinct sides to it. As such, MutLα can only interact with PCNA on a specific face and the complex will have a certain orientation, which remains constant even when sliding across the DNA. MutLα has endonuclease activity on one of its heterodimer subunit, PMS2, and this will only nick the same strand as the PCNA binding.

The reason that nicks are made close by to the mismatch (which is essential for DNA repair) is because the complex making the nicks, MutSα/MutLα, has the highest number around the mismatch site, correlating with greater PCNA collision frequency. This is especially important in replication, where the PCNA molecules adhere to the DNA for an extended period of time even after replication. Due to RFC, they are loaded at the 3' terminus of an Okazaki fragment of the leading strand. They adhere with a certain orientation, which allows MutSα/MutLα to cleave the nascent DNA strand, even though the gap around the Okazaki fragment has long been linked. As such, the MMR system has the correct directionality due to this nick generation.

DNA Repair by DNA ligase I

Degradation

In degradation, EXO1 is loaded at the nicks created by the PCNA-activated MutSα/MutLα complex. This creates a large gap that starts the nick and ends around 150 bases after the mismatch. This gap is single-stranded and on the same side as the mismatch. EXO1 is an exonuclease that can only cut in a 5' to 3' direction.

Resynthesis

Resynthesis involves PCNA, polymerase-δ, and DNA ligase I in order to replace the removed bases and, overall, fix the mismatch error.

EXO1 Independent Mechanism

Although not proven in humans, EXO1 deficient mice showed less mutations than MSH2 and MLH1 deficient mice, indicating a mismatch repair mechanism that does not require EXO1. Indeed, a 5' nick MMR mechanism could occur without EXO1 through use of polymerase-δ and MutSα, RPA, RFC, and PCNA. When there is a 5' nick from the mismatch error, polymerase-δ can catalyze strand displacement, whereby FEN1 can catalyze the removal of the strand containing the mismatch. DNA ligase I would then seal the nick formed.

Insertion/Deletion Loops and Trinucleotide Repeats

Insertion/Deletion loops (IDLs) and trinucleotide repeats (TNRs) interact largely with MMR in both error-preventing and error-propagating ways.

Origin of IDLs

IDLs arise due to the activity of polymerase on TNRs. Trinucleotide repeats are large number of repeats of a single tripley of nucleotides. Such repeats have been implicated in diseases such as Fragile X. When polymerase reads these repeats, it slows down. However, helicase does not slow down, and due to being relatively faster, there becomes long strands of single stranded DNA. As such, these strands can bunch up and form an IDL. This would cause polymerase to create shorter than usual DNA strands.

The Error-Preventing Role of MMR

When things such as this happen, MMR can work to fix it. If the loops is less than two to three extrahelical nucleotides long, the canonical MMR can fix it. However, if the loop is longer, there is a MutSβ-mediated way for loops to be fixed. However, this happens by some other MMR mechanism, for in the regular process, PCNA would not be able to diffuse past the large loop. Thus, there must be some non EXO1-mediated MMR.

The Error-Propagating Role of MMR

In certain cases, MMR may be "hijacked" to cause TNR expansion. In the event that there is a cruciform loop structure, where there are loops in both strands in the same relative position, a cleavage by PCNA attached by RFC onto one of the loops activating MutSβ and MutLα endonucleolytic activity may cause one of the loops to collapse. When polymerase replaces the missing nucleotides, there will be an extension of the trinucleotide repeat. A larger number of repeats has been linked to more severe disease in diseases (such as Huntington's Disease) that are caused by TNRs, and so this is an important field of study.

Antibody Variation and Class-Switching

Antibody variation, although due to a variety of reasons, is largely dependent on the role of MMR. After VDJ recombination, a process involving recombination of the variable, diversity, and join regions of the immunoglobulin genes, a variety of IgM antibodies can be made. However, there are further mechnanisms for antibody variability.

The role of MMR in Somatic Hypermutation

Somatic hypermutation (SHM) is a process whereby many mutations arise in the variable region of the antibody. It works through activation-induced cytidine deaminase (AID) where C nucleotides are converted to U. This occurs during transcription, because AID works best on single-stranded DNA. When this occurs, there is a mismatch error on the resultant DNA. As such, uracil DNA-glycosylase works to do base-excision repair (BER) and remove the incorrect U nucleotide. However, once this happens there is an apyrimidinic site (AP) remaining. This site can be the target of EXO1 in order for MMR to occur.

In this case, EXO1 cleavage may cause a large swath of DNA to be excised. When polymerase goes to fix it, AP sites and remaining uracil nucleotides may cause incorrect mutations at the sites where AID acts. This would result in changes in the variable site of the antibody and ultimately, different antigen recognition.

The role of MMR in Class-Switch Recombination

MMR can also cause the type of antibody to change, such as from IgG to IgM while recognizing the same type of antigen. When there are two AP sites and EXO1 causes the excision of a section of DNA to the other gap, class-switch recombination (CSR) can occur due to a double-strand break.

References

1. Peña-Diaz, J., & Jiricny, J. (2012). Mammalian mismatch repair: error-free or error-prone? Trends in biochemical sciences, 37(5), 206–14. doi:10.1016/j.tibs.2012.03.001
2. Zhao, J. et al. (2009) Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks. Nucleic Acids Res. 37, 4420-4429
3. Lopez Castel, A. et al (2010) Repeat instability as the basis for human diseases and as a potential target for therapy. Nat. Rev. Mol. Cell Biol. 11, 165-170.