Recombinational excision

Figure 4.5. Integrative and excisive X phage recombination pathways. Integration is catalyzed by the X Int protein in a reaction that also requires the E. coli IHF protein. Recombination occurs within a common core sequence of 15 base pairs. The excision reaction requires the X Xis protein in addition to Int and IHF.

The reaction differs from excision of the X chromosome because the Entry Clone contains two attL sites and the destination vector contains two attR sites (Hartley et al., 2000). The att sites are mutated to ensure recombination only occurs between attLl and attRl and between attL2 and att.R2. The recombination reaction proceeds through a cointegrate molecule that is resolved to create a destination vector containing the gene of interest with the desired promoter and tag sequences (Fig. 4.6). 

Marker excision by site-specific recombination Very clean excision, small footprint Complex cloning procedure. Requires additional transgene encoding Cre recombinase 39... 

An alternative method (developed in the Eli Lilly research laboratories), entails inserting a nucleotide sequence coding for human proinsulin into recombinant E. coli. This is followed by purification of the expressed proinsulin and subsequent proteolytic excision of the C peptide in vitro. This approach has become more popular, largely due to the requirement for a single fermentation and subsequent purification scheme. Such preparations have been termed human insulin prb ... 

Mismatch Repair. Mispairs that break the normal base-pairing rules can arise spontaneously due to DNA biosynthetic errors, events associated with genetic recombination and the deamination of methylated cytosine (Modrich, 1987). With the latter, when cytosine deaminates to uracil, an endonuclease enzyme, /V-uracil-DNA glycosylase (Lindahl, 1979), excises the uracil residue before it can pair with adenine at the next replication. However, 5-methyl cytosine deaminates to form thymine and will not be excised by a glycosylase. As a result, thymine exits on one strand paired with guanine on the sister strand, that is, a mismatch. This will result in a spontaneous point mutation if left unrepaired. For this reason, methylated cytosines form spontaneous mutation hot-spots (Miller, 1985). The cell is able to repair mismatches by being able to distinguish between the DNA strand that exists before replication and a newly synthesized strand. 

Fig. 1. Proteins in DNA repair pathways. DNA repair proteins are listed for each of the following pathways BER (Base Excision Repair), NER (Nucleotide Excision Repair), MMR (Mismatch Repair), HR (Homologous Recombination), and NHEJ (Nonhomologous End Joining). PARP1/2 and BRCA1/2 are relevant in BER and HR pathways, respectively.

DNA repair pathways can be divided into those that respond to SSB and those that respond to DSB. SSB repair pathways include base excision repair (BER), mismatch repair (MMR), and nucleotide excision repair (NER). DSB repair pathways include nonhomologous end joining (NHEJ) and homologous recombination (HR). The proteins involved in these DNA repair pathways are shown in Fig. 1. 

The repair of stalled replication forks entails a coordinated transition from replication to recombination and back to replication. The recombination steps function to fill the DNA gap or rejoin the broken DNA branch to recreate the branched DNA structure at the replication fork. Lesions left behind in what is now duplex DNA are repaired by pathways such as base-excision or nucleotide-excision repair. Thus a wide range of enzymes encompassing every aspect of DNA metabolism ultimately take part in the repair of a stalled replication fork. This type of repair process is clearly a primary function of the homologous recombination system of every cell, and defects in recombinational DNA repair play an important role in human disease (Box 25-1). 

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