Gene repair experiment

1Gene repair experimentThe core concept and biological significance of gene repair

Gene repair (DNA Repair) is a molecular mechanism by which cells recognize and correct DNA damage (such as mutations, breaks, chemical modifications), which plays a crucial role in maintainingGenomic stabilitycrucial If there is no mechanism of recombination, the spontaneous mutation rate will increase by more than 1000 times. Its core values include:

1. Defend against genetic errorsPrevent the accumulation of mutations such as point mutations, insertions/deletions, etc.

2. Ensure cellular functionTo avoid cell apoptosis or carcinogenesis caused by DNA damage.

3. Evolutionary balanceBalancing fidelity repair with allowing for moderate variation, supporting adaptive evolution.

IIGene repair experimentClassification and molecular pathways of main repair mechanisms

According to the type of injury and repair strategy, it can be divided into the following five categories:

（1） Mismatch Repair (MMR)

• targetBase mismatches caused by replication errors (such as A-C pairing), single base insertions/deletions.

• key steps：

1. recognizeMutS protein (prokaryotic MutS, eukaryotic MSH2/MSH6) recognizes mismatch sites.

2. Chain differentiationDetermine the chain that needs to be repaired based on the unmethylated state of the newly synthesized chain (prokaryotic) or PCNA labeling (eukaryotic).

3. Removal and repairMutL/ExoI removes the erroneous fragment, and DNA polymerase δ/ε and ligase complete the repair.

• Disease associationMMR deficiency leads to hereditary non polypoid colorectal cancer (HNPCC).

（2） Excision Repair

Divided into three categories based on the extent of damage:

1. Base Excision Repair (BER)

• goalOxidative/alkylated damaged bases (such as 8-oxoniao purine).
  

• process：

• DNA glycosylation enzyme cleaves damaged base → AP site formation → AP endonuclease cleaves phosphodiester bond → DNA polymerase β fills → Ligase closes gap.

2. Nucleotide Excision Repair (NER)

• goalUV induced damage to pyrimidine dimers, chemical adducts, and other large fragments.

• mechanism：

• XPC-RAD23B complex recognizes damage → TFIIH uncoils DNA → XPA/XPG cleaves 24-32 nt fragments containing damage → DNA polymerase δ/ε/κ synthesizes new chains.

• Disease associationNER defects lead to Xeroderma Pigmentosum.

3. Direct Repair

• goalSpecific chemical modifications (such as O ⁶ - methylniao purine).

• Enzyme mediatedMGMT protein directly transfers methyl groups without the need for cleavage.

（3） Double Strand Break Repair (DSBR)

DNA double strand breaks are the most lethal damage, mainly repaired through two pathways:

1. Non Homologous End Joining (NHEJ)

• featureQuick but error prone, connect the broken end directly.

• key proteinKu70/80 recognizes breakage → DNA PKcs activation → XLF/XRCC4/Ligase IV linkage.

• riskEasy to cause insertion/deletion mutations (indel), which is the main cause of off target effects in CRISPR editing.

2. Homologous Recombination (HR)

• feature: High fidelity, requiring sisters chromatids as templates.

• process：

• MRN complex cleaves 5 'end → RAD51 forms single stranded DNA protein filaments → invades homologous template → DNA synthesis → Holliday linker dissociation.

• applicationCRISPR-HDR technology enables precise gene knock in or point mutation repair.

（4） Synthesis Dependent Strand Annealing (SDSA)

• positioningSubtypes repaired by HR to avoid cross recombination.

• mechanism：

• After cutting off the broken end, it invades the homologous template → DNA synthesis and extension → new strand detachment from the template and annealing with the other broken end → connection completion and repair.

• Technical valueThe ExACT model combining CRISPR with single stranded oligonucleotide (ssODN) is based on SDSA, achieving precise repair of point mutations.