The replication process of prokaryotic DNA is briefly explained.
1.unwinding of DNA double helix
In the process of DNA replication, double-stranded DNA is first untied to form replication fork, while the formation of replication fork is a complex replication process, involving a variety of protein and enzymes.
(1) single-stranded DNA binding protein (SSB DNA protein).
SsbDNA protein is a kind of protein that firmly binds to single-stranded DNA. Prokaryote ssbDNA protein shows synergistic effect when binding to DNA: if the binding capacity of the 1 th ssbDNA protein to DNA is 1, the binding capacity of the second SSB DNA protein can reach103; SsbDNA protein in eukaryotic cells does not show the above effect when it binds to single-stranded DNA. The function of ssbDNA protein is to ensure that the single strand of the unwinding enzymatic hydrolysate can maintain its single strand structure until the replication is completed. It exists in replication fork in the form of tetramer, which will be removed and recycled after single-stranded replication. Therefore, ssbDNA protein only maintains the existence of single strand and has no unwinding effect.
(2)DNA helicase
DNA melting enzyme can obtain energy by hydrolyzing ATP to untie double-stranded DNA. The activity of this melting enzyme to decompose ATP depends on the existence of single-stranded DNA. If there is a single-stranded end or gap in double-stranded DNA, DNA melting enzyme can first bind to this part, and then gradually move to the double-stranded direction. In the process of replication, most DNA helicase can move along the 5'-> 3' direction of the lagging template, and with the progress of replication fork, only a few helicase (Rep protein) move along the 3'-> 5' direction. Therefore, it is speculated that Rep protein and specific DNA melting enzyme cooperate to untie double-stranded DNA on two mother chains of DNA.
(3)DNA melting process
Dna is not only a double helix but also a supercoiled state before replication, and the existence of supercoiled state is a necessary structural state before melting. In addition to understanding streptozyme, there are some specific protein, such as DNA protein in E.coli. Once part of double-stranded DNA is unbound, ssbDNA protein must be available to stabilize the unbound single-stranded DNA to ensure that this part will not return to double-stranded. The initial process of replication of two single-stranded DNA is different, but both leading strand and subsequent strands need RNA primers to start the synthesis of sub-stranded DNA. Therefore, the difference between leading strand and the subsequent chain is that the former continues to synthesize 5'-3' from the replication origin without forming Okazaki fragments, while the latter continuously synthesizes Okazaki fragments with a length of about 2-3 KB with the appearance of replication fork.
2. Okazaki fragment and semi-discontinuous replication
Because the two strands of DNA are anti-parallel, one DNA untied near replication fork is in the direction of 5'-> 3', and the other is in the direction of 3'-> 5'. The polarities of the two templates are different. The synthetic direction of all known DNA polymerases is 5'-> 3' direction, not 3'-> 5' direction, so it cannot explain the problem of simultaneous replication of two DNAs. In order to explain the phenomenon of isokinetic replication of each template synthesis subunit of two DNA chains, Japanese scholar Okazaki and others proposed a semi-discontinuous replication model of DNA. In 1968, okazaki labeled Escherichia coli with 3H deoxythymidine for a short time, extracted DNA, and after denaturation, ultracentrifugation obtained many 3H labeled DNA, which was called okazaki fragment. After prolonging the labeling time, Okazaki fragments can be transformed into mature DNA chains, so these fragments must be intermediate products in the replication process. Another experiment also proved that in the process of DNA replication, smaller fragments were synthesized first, that is, the temperature-sensitive mutant of DNA ligase was tested, and a large number of small DNA fragments were accumulated at the temperature where ligase did not work, indicating that at least one strand synthesized shorter fragments first, and then the ligase chain became macromolecular DNA in the process of DNA replication. Generally speaking, the okazaki fragment of prokaryotes is longer than that of eukaryotes. In-depth research has also proved that leading strand's continuous replication and discontinuous replication of lagging chain are common in biology, so they are called semi-discontinuous replication of DNA double helix.
3. Initiation and termination of replication
All DNA replication starts from a fixed starting point. DNA polymerase can only extend the existing DNA chain, but can't synthesize DNA chain from scratch. How is the replication of new DNA formed? A large number of experimental studies have proved that in DNA replication, RNA polymerase usually synthesizes an RNA primer on the DNA template, and then polymerase synthesizes a new DNA strand from the 3' end of the RNA primer. For the main chain, this initiation process is relatively simple. As long as there is an RNA primer, DNA polymerase can be synthesized from it. For the following chain, the initial process is complicated, which requires a variety of protein and enzymes. The initiation process of the subsequent chain is completed by the initiator. The initiator consists of six kinds of protein, and the six kinds of protein are combined by pre-initiator or precursor, and further assembled with initiating enzyme or primer processing enzyme to form initiator. Like a locomotive, the initiator moves along the direction of chain bifurcation, intermittently triggering the generation of short primer RNA chains with lagging chains on the template, and then synthesizing DNA under the action of DNA polymerase III until it meets the next primer or Okazaki fragment. RNA primers were degraded by RNase H, gaps were filled by DNA polymerase I, and then every two Okazaki fragments were linked together by DNA ligase to form macromolecular DNA.
(4) Telomere and telomerase
194 1 year, the American Indian Mc Clintock put forward the telomere hypothesis, and thought that there must be a special structure at the end of chromosome-telomere. At present, it is known that chromosome telomeres have at least two functions: ① to protect chromosome ends from damage and maintain chromosome stability; ② It is connected with the nuclear fiber layer, so that the chromosome can be located.
After understanding the process of DNA replication, scientists questioned how the gap was filled after the RNA primer at the 5' end of the new strand was removed during DNA replication in the 1970s. If you don't fill it in, the DNA will get shorter every time you copy it. For example, when RNA primers are removed, Okazaki fragments are filled with DNA synthesized by DNA polymerase I, and then they are connected into a complete chain by DNA ligase. However, when DNA polymerase I catalyzes DNA synthesis, it needs free 3'-OH as primer, and the 5' of the remaining sub-chain can't be filled, so the chromosome is a little short.
In normal somatic cells, it is common that telomeres will shorten once chromosomal enzymes are copied. It is speculated that once telomeres are shortened to a certain threshold length, an alarm will be issued, indicating that cells are aging; Perhaps when a cell judges that its chromosome becomes too short, division stops, which leads to a certain limitation of the life span of normal somatic cells. However, in cancer cells, telomeres of chromosomes keep a certain length. Why? This is because after DNA replication, the short part at the end of chromosome is supplemented with telomerase, an enzyme containing RNA, which not only solves the problem of template but also solves the problem of primer. Telomerase activity was detected in germ cells and 85% cancer cells, but not in normal somatic cells. In the mid-1990s, Blackburn cloned the telomerase gene in protozoa for the first time.
Telomerase is active in cancer cells, which can not only make cancer cells divide and proliferate continuously, but also provide time for cells before or after cancer to accumulate additional mutations, which is equivalent to increasing their ability to replicate, invade and eventually metastasize. At the same time, people have developed drugs for telomeres, that is, to treat cancer by inhibiting telomerase activity in cancer cells.
As for the structural characteristics of the DNA end of eukaryotic cells, as early as 1978, Blackburn took the emergence of protozoan four membranes (a kind of ciliates) as an example: ① zigzag hairpin ring; ② Simple sequence (C4 A2) consisting of only C and A repeats (20 ~ 70); There are many nicks on the chain.