Weaver/Molecular Biology

Excision Repair in Prokaryotes

The percentage of DNA damage products that can be handled by direct reversal is necessarily small. Most such damage products involve neither pyrimidine dimers nor O6-alkylguanine, so they must be handled by a different mechanism. Most are removed by a process called excision repair. The damaged DNA is first removed, then replaced with fresh DNA. In E. coli this occurs by one of two mechanisms: base excision repair or nucleotide excision repair.

Base Excision Repair

In base excision repair, bulky damage is recognized by an enzyme called DNA glycosylase, which breaks the glycosidic bond between the damaged base and its sugar (Figure 6). This leaves an apurinic or apyrimidinic site (AP site), which is a sugar without its purine or pyrimidine base. Once the AP site is created, it is recognized by AP endonucleases that cut, or nick, the DNA strand on either side of the AP site; removing the AP sugar-phosphate. (The "endo" in endonuclease means the enzyme cuts inside a DNA strand, not at a free end; Greek: endo = within.) DNA polymerase fills the resulting gap by inserting a nucleotide to pair with the one in the opposite strand. DNA ligase seals the remaining nick to complete the job.

Nucleotide Excision Repair

Bulky base damage, including thymine dimers, can also be removed directly, without help from a DNA glycosylase. In this nucleotide excision repair (NER) pathway (Figure 7), the incising enzyme system makes cuts on either side of the damage, removing an oligonucleotide with the damage. The key enzyme E. coli cells use in this process is called the uvrABC endonuclease because it contains three polypeptides, the products of the uvrA, uvrB, and uvrC genes. This enzyme generates an oligonucleotide that is twelve to thirteen bases long, depending on whether the damage affects one nucleotide (alkylations) or two (thymine dimers). A more general term for the enzyme system that catalyzes nucleotide excision repair is excision nuclease, or excinuclease. As we will see later in this chapter, the excinuclease in eukaryotic cells removes an oligonucleotide about 24 to 32 nucleotides long, rather than a 12- to 13-mer. In any case, DNA polymerase fills in the gap left by the excised oligonucleotide and DNA ligase seals the final nick.

Summary: Most DNA damage in E. coli is corrected by excision repair, in which the damaged DNA is removed and replaced with normal DNA. This can occur by two different mechanisms. (1) The damaged base can be clipped out by a DNA glycosylase, leaving an apurinic or apyrimidinic site that attracts the DNA cutting enzymes that remove the damaged region. (2) The damaged DNA can be clipped out directly by cutting on both sides with an endonuclease to remove the damaged DNA as part of an oligonucleotide. DNA polymerase I fills in the gap and DNA ligase seals the final nick.

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