Chimeric Polymerase SSB Fusion for DNA Replication Processivity
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Solution Overview
Problem
DNA polymerases and single-stranded DNA binding proteins (SSBs) have weak interactions, leading to transient associations that hinder processivity during DNA replication, especially in navigating through secondary structures.
Innovation Solution
A chimeric protein is created by joining a sequence nonspecific single-stranded nucleic-acid-binding domain with a catalytic nucleic-acid-modifying domain, enhancing their affinity and stability, thereby preventing separation and improving processivity while maintaining fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA polymerase and SSB are used as separate proteins, then the system is simpler and easier to manufacture, but the processivity is low due to weak transient interactions
Solution Approach 1:
The patent merges the DNA polymerase and SSB into a single chimeric protein by fusing the polymerase catalytic domain with the SSB oligonucleotide-binding domain. This fusion creates a unified structure that eliminates the need for weak transient interactions between separate proteins, thereby significantly improving processivity while maintaining manufacturing simplicity
Solution Approach 2:
The chimeric protein represents a composite molecular structure combining two functional domains (polymerase and SSB) into one hybrid protein. This composite approach allows the molecule to simultaneously exhibit DNA synthesis capability and enhanced processivity through its built-in SSB domain that binds tightly to the polymerase
2Reliability
If DNA polymerase interacts with accessory proteins like SSB, then processivity increases, but the interaction is weak and transient which hinders sustained replication
Solution Approach 1:
By fusing SSB and DNA polymerase into a single chimeric protein, the invention eliminates the weak transient interactions between separate proteins. The covalent bond within the chimeric structure ensures stable and sustained interaction between the SSB domain and polymerase domain, maintaining processivity without the limitations of transient binding
Solution Approach 2:
The chimeric protein structure acts as an intermediary that bridges the functions of SSB and DNA polymerase. The fused domains work as an integrated unit where the SSB domain continuously engages the polymerase domain, providing stable processivity enhancement without relying on weak external protein-protein interactions
3Reliability
If the SSB binds tightly to ssDNA, then it protects DNA from nucleases, but it may hinder DNA polymerase access to the template
Solution Approach 1:
The chimeric protein merges the SSB's DNA-binding function with the polymerase's catalytic function into a single coordinated unit. The SSB domain binds to ssDNA while the polymerase domain simultaneously accesses the template through this controlled binding, resolving the conflict between DNA protection and polymerase access
Solution Approach 2:
The chimeric protein exhibits local quality differentiation where the SSB domain binds tightly to protect ssDNA regions while the polymerase domain maintains access to the template strand. This spatial separation of functions within the single protein allows simultaneous DNA protection and efficient replication
Data Source
AI summary
Chimeric proteins comprising a sequence nonspecific single-stranded nucleic-acid-binding domain joined to a catalytic nucleic-acid-modifying domain are provided. Methods comprising contacting a nucleic acid molecule with a chimeric protein, as well as systems comprising a nucleic acid molecule, a chimeric protein, and an aqueous solution are also provided. The joining of sequence nonspecific single-stranded nucleic-acid-binding domain and a catalytic nucleic-acid-modifying domain in chimeric proteins, among other things, may prevent the separation of the two domains due to their weak association and thereby enhances processivity while maintaining fidelity.