Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies. Kenneth N Kreuzer
two copies of the core polymerase, and so the leading- and lagging-strand polymerases are physically coupled to each other during replication. As mentioned earlier, the clamp loader plays a key role in this coupling, with two of its subunits binding the polymerase on the leading and the lagging strand.
While the two polymerases are clearly coordinated with each other, they are apparently not as tightly coupled as in the bacteriophage T7 system. Under at least some conditions, blockage of the lagging-strand polymerase does not cause the leading-strand polymerase to stall. Instead, the leading-strand polymerase and helicase continue onward, generating a longer patch of ssDNA on the lagging-strand template (Figure 3.4A). This in turn can lead to release of the blocked lagging-strand polymerase so that it can eventually cycle to a new RNA primer and restart Okazaki fragment synthesis (Figure 3.4B). Imagine that a damaged template base initially blocked the lagging-strand polymerase. This series of events would lead to a patch of single-stranded template DNA adjacent to the damaged template base, but the fork would continue onward and replication of the rest of the chromosome could be completed. Some further DNA repair reaction would be needed to deal with the small unreplicated patch (and the blocking lesion), but the cell would be able to complete chromosomal replication and proceed with cell division. We will discuss additional pathways for completing replication in unusual situations in the section on replication restart below.
Figure 3.4.Response of the Escherichia coli replication complex to blocking damage on the lagging-strand template. The replication machinery is capable of bypassing blocking damage on the lagging-strand template. The leading-strand polymerase continues onward even though the lagging-strand polymerase is blocked (A). After some delay, the lagging-strand polymerase core complex blocked by damage dissociates from the site of blockage and engages a new RNA primer to resume Okazaki fragment synthesis (B), leaving behind a patch of ssDNA downstream of the blocking lesion.
3.5The E. coli ssDNA-binding protein
The E. coli ssDNA-binding protein is required for normal replication and to protect ssDNA that is generated during replication and other cellular processes. The protein is a tetramer that consists of four identical subunits, each with an N-terminal domain that binds ssDNA and a C-terminal domain that binds a variety of cellular proteins. Like the phage T7 ssDNA-binding protein discussed in Chapter 2, the region of the E. coli ssDNA-binding protein that binds ssDNA consists of an OB-fold (oligonucleotide/oligosaccharide-binding fold).
The bacterial ssDNA-binding protein plays a crucial role during DNA replication. It rapidly and efficiently binds the single-stranded regions exposed on the lagging strand as replication proceeds (Figure 3.2). This binding has a number of important consequences. First, ssDNA is rather sensitive to chemical modifications and breakage, and ssDNA-binding protein protects from these damages. Second, ssDNA can form secondary structures when two segments happen to have complementary sequences, and these secondary structures can be very serious impediments to replication and compromise DNA stability; ssDNA-binding protein prevents the formation of such secondary structures. Third, the protein is thought to help maintain the proper configuration of ssDNA during the rapid reorganizations that occur in the lagging-strand gymnastics of the trombone model.
While E. coli ssDNA-binding protein has all those important functions, it does much more than that. As mentioned earlier, the C-terminus of the protein binds various other proteins and thereby helps to coordinate protein functions for DNA replication. Two key interactions are with primase and with a subunit of the DNA polymerase holoenzyme complex. During the primer handoff mentioned earlier, ssDNA-binding protein is part of the RNA primer–primase complex, and it switches its interaction from primase to the holoenzyme subunit to complete the handoff. ssDNA-binding protein also interacts with a number of proteins involved in repair reactions and replication restart, and we will see in subsequent chapters that the corresponding eukaryotic protein is also central in both DNA replication and repair.
3.6Housekeeping after the replisome passes — Repairing Okazaki fragments, reducing replicative errors, and recycling clamps from the DNA
While the replisome is an efficient machine, several problems remain to be solved after it passes. The most obvious is that the lagging-strand DNA is not complete, but instead is left with multiple embedded RNA primers and discontinuities at the 5′ ends of these primers. As in the T7 system, the RNA primers need to be excised by a 5′ to 3′ exonuclease, replacement DNA bases need to be inserted, and the final nicks in the DNA need to be sealed. At the beginning of this chapter, we mentioned bacterial DNA polymerase I, which was actually the first DNA polymerase purified and characterized (resulting in the 1959 Nobel Prize for Arthur Kornberg). It turns out that this DNA polymerase plays a central role in Okazaki fragment processing. It was initially quite surprising to find that DNA polymerase I contained two exonuclease activities. As with DNA polymerase III, polymerase I has a 3′ to 5′ exonuclease that can edit out mistakes in which the polymerase activity inserted an incorrect base. It also has a 5′ to 3′ exonuclease, and this is the activity that normally removes the RNA primers from Okazaki fragments. Indeed, DNA polymerase I can carry out a concerted reaction called “nick translation,” in which it removes bases from in front of the enzyme with the 5′ to 3′ exonuclease and inserts new bases behind with the DNA polymerase activity (Figure 3.5; note that the position of the nick is “translating” along the DNA). Nick translation efficiently removes the short stretch of RNA at the 5′ ends of Okazaki fragments while replacing the RNA bases with DNA. If any bases are misincorporated during this replacement reaction, the incorrect base will very likely be removed by the proofreading 3′ to 5′ exonuclease activity of polymerase I before completion of the replacement reaction. The 5′ to 3′ exonuclease activity of DNA polymerase I is in a distinct domain of the protein, which can be released from the enzyme by proteolysis.3
1959 Nobel Prize in Physiology or Medicine
This prize was awarded jointly to Severo Ochoa and Arthur Kornberg for their studies on RNA (Ochoa) and DNA (Kornberg) synthesis; Kornberg isolated DNA polymerase from bacteria and demonstrated its ability to synthesize a complementary strand of DNA using a template strand. See legend to Figure 2.1 for details and attribution of DNA polymerase structure.
https://www.nobelprize.org/prizes/medicine/1962/summary/
Figure 3.5. Role of DNA polymerase I in final editing of Okazaki fragments. Escherichia coli DNA polymerase I has both 5′ to 3′ and 3′ to 5′ exonuclease activities, residing in different domains of the protein (panel A). The coordinated action of the 5′ to 3′ exonuclease and the (5′ to 3′) DNA polymerase is referred to as nick translation (panel B), since the location of the nick in the DNA is moving along the DNA. After the RNA residues are all removed, DNA ligase seals the nick to complete lagging-strand synthesis (not shown).
Once the RNA segments are replaced with DNA and the nick has migrated accordingly, the nick is sealed by bacterial DNA ligase. E. coli DNA ligase also plays critical roles in DNA repair reactions, as we will see later in the book.
The DNA synthesized by either DNA polymerase III or DNA polymerase I has very few remaining errors, thanks to the proofreading activities of these enzymes as described earlier. Nonetheless, without further corrective mechanisms, E. coli cells would suffer on the order of one mutation during every round of DNA replication. Measurements of mutation rates