Question : How would and could natural , unguided processes have figured out 1. the requirement of high-speed of polymerization ? How could they have figured out the right configuration and process to do so ? how could natural processes have emerged with the right proteins incrementally, with the hand-shaped clamp loader, and the precisely fitting clamp , enabling the fast process ?? Even the most intelligent scientists are still not able to imagine how this process is engineered ? Furthermore, the process requires molecular energy in the form of ATP, and everything must fit together, and be functional. Without the clamp loader protein, the clamp could not be positioned to the polymerase enzyme, and processivity would not rise to the required speed. The whole process must also be regulated and controlled. How could that regulation have been programmed ? Trial and error ?
Several Proteins Are Required for DNA Replication at the Replication Fork
http://reasonandscience.heavenforum....okaryotes#4398
The various proteins involved in DNA replication are all closely associated in one large complex, called a replisome.
Leading strand synthesis: On the template strand with 3’-5’ orientation, new DNA is made continuously in 5’-3’ direction towards the replication fork. The new strand that is continuously synthesized in 5’-3’ direction is the leading strand.
Lagging strand synthesis: In the lagging strand, the synthesis of DNA also elongates in a 5ʹ to 3ʹ manner, but it does so in the direction away from the replication fork. In the lagging strand, RNA primers must repeatedly initiate the synthesis of short segments of DNA; thus, the synthesis has to be discontinuous.
The Primase (DnaG) enzyme, and the primosome complex
http://reasonandscience.heavenforum....okaryotes#4379
The length of these fragments in bacteria is typically 1000 to 2000 nucleotides. In eukaryotes, the fragments are shorter—100 to 200 nucleotides. Each fragment contains a short RNA primer at the 5ʹ end, which is made by primase. The remainder of the fragment is a strand of DNA made by DNA polymerase III. The DNA fragments made in this manner are known as Okazaki fragments. To complete the synthesis of Okazaki fragments within the lagging strand, three additional events must occur: removal of the RNA primers, synthesis of DNA in the area where the primers have been removed, and the covalent attachment of adjacent fragments of DNA. In E. coli, the RNA primers are removed by the action of DNA polymerase I. This enzyme has a 5ʹ to 3ʹ exonuclease activity, which means that DNA polymerase I digests away the RNA primers in a 5ʹ to 3ʹ direction, leaving a vacant area. DNA polymerase I then synthesizes DNA to fill in this region. It uses the 3ʹ end of an adjacent Okazaki fragment as a primer. , DNA polymerase I would remove the RNA primer from the first Okazaki fragment and then synthesize DNA in the vacant region by attaching nucleotides to the 3ʹ end of the second Okazaki fragment. After the gap has been completely filled in, a covalent bond is still missing between the last nucleotide added by DNA polymerase I and the adjacent DNA strand that had been previously made by DNA polymerase III. To the left of the origin, the top strand is made continuously, whereas to the right of the origin it is made in Okazaki fragments. By comparison, the synthesis of the bottom strand is just the opposite. To the left of the origin it is made in Okazaki fragments and to the right of the origin the synthesis is continuous. Finally the two ends of the fragment have to be joined together; this is the job of an enzyme called DNA ligase. After the completion of one Okazaki fragment , the equipment has to be released, the clamp has to let go, and a new clamp has to be loaded at the beginning of the next fragment. Clearly the formation and control of the replication fork is an enormously complex process.
Step 5: After DNA synthesis by DNA pol III, DNA polymerase I uses its 5’-3’ exonuclease activity to remove the RNA primer and fills the gaps with new DNA. In the next step, finally DNA ligase joins the ends of the DNA fragments together. As the replisome moves along the DNA in the direction of the replication fork, it must accommodate the fact that DNA is being synthesized in opposite directions along the template on the two stands. Picture above provides a schematic model illustrating how this might be accomplished by folding the lagging strand template into a loop.Creating such a loop allows the DNA polymerase molecules on both the leading and lagging strands to move in the same physical direction, even though the two template strands are oriented with opposite polarity. The replisome faces special challenges as it makes new DNA at rates that can approach 1,000 nucleotides per second. Unlike the machines that make proteins and RNA, which work relatively sluggishly and in a linear fashion, the replisome must simultaneously copy two strands of DNA that are aligned in opposite directions (5ʹ to 3ʹ and 3ʹ to 5ʹ). Replisome chemistry obeys two rules.
Questions: How did they arise with that cabability to " obey two rules " ? Suppose a primitive polymerase were duplicated and somehow started to replicate the second strand in the opposite direction while remaining attached to the first strand - how could that change have been directed , and why should that feat have happened randomly ?
The DNA polymerase holoenzyme alone would not be able to duplicate the long DNA faithfully. Tests have shown that Polymerase III alone gets stuck. Furthermore, Polymerase III is not a simple enzyme. Its rather three enzymes in one. Beside replicating DNA, it can also degrade DNA in two different ways. It does so by three different, discrete regions of the molecule. The exonuclease activity plays a critical role in replication. It allows the enzyme to proofread the new DNA and cut out any mistakes it has made. Although the polymerase reads the sequence of the old DNA to produce a new DNA, it turns out that simple base bairing allows about one mistake per thousand base pairs copied. Proofreading reduces errors to about one mistake in a million base pairs. The question is if wheter a proofreading exonuclease and other DNA repair mechanisms had to be present in the very first cell.
Eigen’s theory revealed the existence of the fundamental limit on the fidelity of replication (the Eigen threshold): If the product of the error (mutation) rate and the information capacity (genome size) is below the Eigen threshold, there will be stable inheritance and hence evolution; however, if it is above the threshold, the mutational meltdown and extinction become inevitable (Eigen, 1971). The Eigen threshold lies somewhere between 1 and 10 mutations per round of replication (Tejero, et al., 2011) regardless of the exact value, staying above the threshold fidelity is required for sustainable replication and so is a prerequisite for the start of biological evolution. Indeed, the very origin of the first organisms presents at least an appearance of a paradox because a certain minimum level of complexity is required to make self-replication possible at all; high-fidelity replication requires additional functionalities that need even more information to be encoded (Penny, 2005). The crucial question in the study of the origin of life is how the Darwin-Eigen cycle started—how was the minimum complexity that is required to achieve the minimally acceptable replication fidelity attained? In even the simplest modern systems, such as RNA viruses with the replication fidelity of only about 10^3 and viroids that replicate with the lowest fidelity among the known replicons (about 10^2; Gago, et al., 2009), replication is catalyzed by complex protein polymerases. The replicase itself is produced by translation of the respective mRNA(s), which is mediated by the immensely complex ribosomal apparatus. Hence, the dramatic paradox of the origin of life is that, to attain the minimum complexity required for a biological system to start on the Darwin-Eigen spiral, a system of a far greater complexity appears to be required. How such a system could evolve is a puzzle that defeats conventional evolutionary thinking, all of which is about biological systems moving along the spiral; the solution is bound to be unusual.
DNA damage and repair
http://reasonandscience.heavenforum....ght=dna+repair
http://reasonandscience.heavenforum....okaryotes#4401
Replication forks may stall frequently and require some form of repair to allow completion of chromosomal duplication. Failure to solve these replicative problems comes at a high price, with the consequences being genome instability, cell death and, in higher organisms, cancer. Replication fork repair and hence reloading of DnaB may be needed away from oriC at any point within the chromosome and at any stage during chromosomal duplication. The potentially catastrophic effects of uncontrolled initiation of chromosomal duplication on genome stability suggests that replication restart must be regulated as tightly as DnaA-directed replication initiation at oriC. This implies reloading of DnaB must occur only on ssDNA at repaired forks or D-loops rather than onto other regions of ssDNA, such as those created by blocks to lagging strand synthesis.Thus an alternative replication initiator protein, PriA helicase, is utilized during replication restart to reload DnaB back onto the chromosome